Method of detecting heat-resistant fungus

ABSTRACT

A method of detecting a heat-resistant fungus, which has a step of identifying the heat-resistant fungus using the following nucleic acid (I) or (II):
         (I) a nucleic acid including a nucleotide sequence set forth in any one of SEQ ID NOS: 24 to 35 and 83 to 86, or a complementary sequence thereof; or   (II) a nucleic acid including a nucleotide sequence resulting from a deletion, substitution, or addition of one to several nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOS: 24 to 35 and 83 to 86 and being capable of detecting the heat-resistant fungus, or a complementary sequence thereof.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing, file name2537 0420001_SeqListing_ascii.txt, size 28,020 bytes; and date ofcreation Jun. 23, 2015, filed herewith, is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a method of detecting a heat-resistantfungus.

BACKGROUND ART

Heat-resistant fungi are widely distributed throughout nature, and thefungi grow proliferously in agricultural crops such as vegetables andfruits and contaminate foods and drinks made from the agriculturalcrops. Moreover, the heat-resistant fungi have high heat resistancecompared with general other fungi. For example, the heat-resistant fungimay survive and grow proliferously even after a heat sterilizationtreatment of an acidic drink and may cause mold growth. Therefore, thereare concerns about the heat-resistant fungi as important harmful fungicausing severe accidents.

As major heat-resistant fungi causing contamination accidents, which maybe detected from foods and drinks after a heat sterilization treatment,heat-resistant fungi belonging to the genera Byssochlamys, Talaromyces,Neosartorya, and Hamigera are known. Compared with other heat-resistantfungi which form ascospores, the fungi belonging to the above-mentionedfour genera have very high heat resistance and are likely to surviveafter heat sterilization. On the other hand, heat-resistant fungi otherthan the above-mentioned four genera can be killed under usualsterilization conditions and hence are less likely to causecontamination accidents unless sterilization fails. Therefore, toprevent the accidents by such heat-resistant fungi in foods and drinksand raw materials thereof, it is particularly important to detect anddiscriminate the heat-resistant fungi belonging to the four genera.

Moreover, to perform accident cause investigation and countermeasure inthe case of a harmful accident, it is necessary to identify a funguscausing the accident. Therefore, if the heat-resistant fungi of theabove-mentioned four genera can be discriminated, the fungus causing theaccident can be detected and discriminated more rapidly.

As a conventional method of detecting and discriminating heat-resistantfungi, a method involving culturing a sample in PDA medium or the likeand detecting fungi is known. However, in this method, it takes aboutseven days until colonies are confirmed. Moreover, identification of thespecies of the fungi is performed based on the morphology of the fungalorgan characteristic to each fungus, and hence it is necessary tocontinue the culture for further seven days until morphologicalcharacters appear. Therefore, according to the method, it takes forabout 14 days to detect and discriminate heat-resistant fungi. Suchmethod which requires a long period of time to detect and discriminateheat-resistant fungi is not necessarily satisfactory in terms ofsanitary management of foods and drinks, freshness keeping of rawmaterials, and distribution constraint. Therefore, it is required toestablish a method of detecting and discriminating heat-resistant fungimore rapidly.

As a method of rapidly detecting and discriminating fungi, detectionmethods using a polymerase chain reaction (PCR) are known (e.g., seePatent Documents 1 to 4). However, such methods have problems in that itis difficult to detect specific heat-resistant fungi specifically andrapidly.

PRIOR ART DOCUMENT Patent Document

[Patent Document 1] JP-T-11-505728 (“JP-T” means published Japanesetranslation of PCT application)

[Patent Document 2] JP-A-2006-61152 (“JP-A” means unexamined publishedJapanese patent application)

[Patent Document 3] JP-A-2006-304763

[Patent Document 4] JP-A-2007-174903

SUMMARY OF INVENTION

The present invention is to provide a method of specifically and rapidlydetecting and discriminating a heat-resistant fungus which is a mainfungus causing contamination of foods and drinks.

The difficulty in detection of a heat-resistant fungi as described aboveis caused by false positive and false negative results in the PCR methodusing known conventional primers.

In view of such problems, the inventors of the present invention havemade extensive studies to search a novel DNA region capable ofspecifically detecting and discriminating the specific heat-resistantfungi. As a result, the inventors have found out that the β-tubulin geneor the ITS region and D1/D2 region of 28S rDNA of the heat-resistantfungi includes a region having a specific nucleotide sequence which canbe clearly different from that of another fungus (hereinafter, alsoreferred to as “variable region”). Moreover, the inventors have foundout that such the heat-resistant fungi can be detected specifically andrapidly by targeting the variable region. The present invention has beencompleted based on the findings.

According to the present invention, there is provided the followingmeans:

The present invention resides in a method of detecting a heat-resistantfungus selected from the group consisting of fungi belonging to thegenus Byssochlamys, fungi belonging to the genus Talaromyces, fungibelonging to the genus Neosartorya, Aspergillus fumigates, and fungibelonging to the genus Hamigera, which has at least one step selectedfrom the group consisting of the following steps 1) to 4):

1) a step of identifying a fungus belonging to the genus Byssochlamysusing the following nucleic acid (A-I) or (A-II):

(A-I) a nucleic acid including a nucleotide sequence set forth in SEQ IDNO: 24 or 25, or a complementary sequence thereof; or

(A-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in SEQ ID NO: 24 or 25 and being capableof detecting the fungus belonging to the genus Byssochlamys, or acomplementary sequence thereof;

2) a step of identifying a fungus belonging to the genus Talaromycesusing the following nucleic acid (B-I) or (B-II):

(B-I) a nucleic acid including a nucleotide sequence set forth in anyone of SEQ ID NOS: 26 to 31, or a complementary sequence thereof; or

(B-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in any one of SEQ ID NOS: 26 to 31 andbeing capable of detecting the fungus belonging to the genusTalaromyces, or a complementary sequence thereof;

3) a step of identifying a fungus belonging to the genus Neosartoryaand/or Aspergillus fumigatus using the following nucleic acid (C-I) or(C-II):

(C-I) a nucleic acid including a nucleotide sequence set forth in anyone of SEQ ID NOS: 32 to 34 and 83 to 86, or a complementary sequencethereof; or

(C-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in any one of SEQ ID NOS: 32 to 34 and 83to 86 and being capable of detecting the fungus belonging to the genusNeosartorya and/or Aspergillus fumigatus, or a complementary sequencethereof; and

4) a step of identifying a fungus belonging to the genus Hamigera usingthe following nucleic acid (D-I) or (D-II):

(D-I) a nucleic acid including a nucleotide sequence set forth in SEQ IDNO: 35, or a complementary sequence thereof; or

(D-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in SEQ ID NO: 35 and being capable ofdetecting the fungus belonging to the genus Hamigera, or a complementarysequence thereof.

Further, the present invention resides in a nucleic acid represented bythe following (I) or (II) for detecting a heat-resistant fungus:

(I) a nucleic acid including a nucleotide sequence set forth in any oneof SEQ ID NOS: 24 to 35 and 83 to 86, or a complementary sequencethereof; or

(II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in any one of SEQ ID NOS: 24 to 35 and 83to 86 and being capable of detecting the heat-resistant fungus, or acomplementary sequence thereof.

Further, the present invention resides in an oligonucleotide fordetecting a heat-resistant fungus, which is capable of hybridizing withthe nucleic acid (I) or (II) described above and has a function as anucleic acid probe or nucleic acid primer for specifically detecting theheat-resistant fungus.

Moreover, the present invention resides in a kit for detecting aheat-resistant fungus containing the above-mentioned oligonucleotidesfor detection as a nucleic acid probe or a nucleic acid primer.

According to the present invention, it is possible to provide a methodof specifically and rapidly detecting and discriminating aheat-resistant fungus which is a main fungus causing contamination offoods and drinks.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram for comparing partial nucleotide sequences of theβ-tubulin genes of Aspergillus fumigatus (SEQ ID NO:34 bases 1-99 and200-299), Neosartorya fischeri fischeri bases 1-97 (SEQ ID NO: 83) andbases 198 to 295 (SEQ ID NO: 84), and Neosartorya fischeri spinosa bases1-100 (SEQ ID NO: 85) and bases 201 to 299 (SEQ ID NO: 86).

FIG. 2 is a diagram illustrating nucleotide sequences of the β-tubulingenes of Hamigera avellanea (SEQ ID NO:35) and Cladosporiumcladosporoides (SEQ ID NO:87).

FIG. 3 is an electrophoretogram showing discrimination results of fungibelonging to the genus Byssochlamys in Example 1(A-1). FIG. 3(a) showsan electrophoretogram of samples of the fungi shown in Table 1, and FIG.3(b) shows an electrophoretogram of samples of the fungi shown in Table2. The numbers in the electrophoretograms correspond the sample numbersin the tables, and represent samples obtained by using DNAs extractedfrom the fungi having the corresponding sample numbers in the tables.

FIG. 4 is an electrophoretogram in the case of using strains ofByssochlamys fulva in Example 1(A-2).

FIG. 5 is an electrophoretogram in the case of using strains ofByssochlamys nivea in Example 1(A-2).

FIG. 6 is an electrophoretogram showing discrimination results of fungibelonging to the genus Talaromyces in Example 1(B-1).

FIG. 7 is an electrophoretogram showing discrimination results of fungibelonging to the genus Talaromyces in Example 1(B-2). FIG. 7(a) shows anelectrophoretogram of samples of the fungi shown in Table 4, and FIG.7(b) shows an electrophoretogram of samples of the fungi shown in Table5. The numbers in the electrophoretograms correspond the sample numbersin each table, and represent samples obtained by using DNAs extractedfrom the fungi having the corresponding sample numbers in the tables.

FIG. 8 is an electrophoretogram showing discrimination results of fungibelonging to the genus Talaromyces in Example 1(B-2).

FIG. 9-1 is an electrophoretogram in Example 1(B-3).

FIG. 9-2 is an electrophoretogram in Example 1(B-4).

FIG. 10 is an electrophoretogram in the case of using strains ofTalaromyces flavus in Example 1(B-5).

FIG. 11 is an electrophoretogram in the case of using strains ofTalaromyces macrosporus in Example 1(B-5).

FIG. 12 is an electrophoretogram showing discrimination results of fungibelonging to the genus Neosartorya and Aspergillus fumigatus in Example1(C-1).

FIG. 13 is an electrophoretogram showing discrimination results of fungibelonging to the genus Neosartorya and Aspergillus fumigatus in Example1(C-2). FIG. 13(a) shows an electrophoretogram of samples of the fungishown in Table 8, and FIG. 13(b) shows an electrophoretogram of samplesof the fungi shown in Table 9. The numbers in the electrophoretogramscorrespond the sample numbers in the tables, and represent samplesobtained by using DNAs extracted from the fungi having the correspondingsample numbers in the tables.

FIG. 14 is an electrophoretogram showing discrimination results ofAspergillus fumigatus from fungi belonging to the genus Neosartorya andAspergillus fumigatus in Example 1(C-3).

FIG. 15 is an electrophoretogram showing discrimination results ofAspergillus fumigatus from fungi belonging to the genus Neosartorya andAspergillus fumigatus in Example 1(C-3).

FIG. 16 is an electrophoretogram in the case of using strains ofNeosartorya fischeri fischeri in Example 1(C-4). FIGS. 16 (a) and (b)show electrophoretograms of samples of strains of Neosartorya fischerifischeri. FIG. 16(a): Primers N2F/R represented by the nucleotidesequences set forth in SEQ ID NOS: 14 and 15 were used. FIG. 16(b):Primers Af1F/R represented by the nucleotide sequences set forth in SEQID NOS: 22 and 23 were used. The following strains were used in each ofFIGS. 16(a) and (b): lane 1: N. fisheri CBM-FA-839 96-PE-92-1; lane 2:N. fisheri CBM-FA-181 90-BP-127-1; lane 3: N. fisheri XY-243-1; lane 4:N. fisheri 2000-XY-247-3; lane 5: N. fisheri XY-296-1; and lane 6: N.fisheri XY-298-1. “P” was the positive control: A. fumigatus strainIFM77.

FIG. 17 is an electrophoretogram in the case of using strains ofNeosartorya fischeri glabra in Example 1(C-4). FIGS. 17 (a) and (b) showelectrophoretograms of samples of strains of Neosartorya glabra. FIG.17(A): Primers N2F/R represented by the nucleotide sequences set forthin SEQ ID NOS: 14 and 15 were used. FIG. 17(b): Primers Af1F/Rrepresented by the nucleotide sequences set forth in SEQ ID NOS: 22 and23 were used. The following strains were used in each of FIGS. 17(a) and(b): lane 1: N. glabra CBM-FA-665 93-BS590-3; lane 2: N. glabraCBM-FA-657 93-BS-1164-3; lane 3: N. glabra CBF158; lane 4: N. glabraCBM-FA-189; lane 5: N. glabra IFM47148; lane 6: N. glabra IFM53839; lane7: N. glabra IFM53841 and lane 8: N. glabra IFM55914. “P” was thepositive control: A. fumigatus strain IFM77.

FIG. 18 is an electrophoretogram in the case of using strains ofNeosartorya hiratsukae in Example 1(C-4). FIGS. 18 (a) and (b) showelectrophoretograms of samples of strains of Neosartorya hiratsukaefischeri. FIG. 18(a): Primers N2F/R represented by the nucleotidesequences set forth in SEQ ID NOS: 14 and 15 were used. FIG. 18(b):Primers Af1F/R represented by the nucleotide sequences set forth in SEQID NOS: 22 and 23 were used. The following strains were used in each ofFIGS. 18(a) and (b): lane 1: N. hiratsukae IFM46953; lane 2: N.hiratsukae 46954; lane 3: N. hiratsukae 47036; lane 4: N. hiratsukae49776; and lane 5: N. hiratsukae 55915. “P” was the positive control: A.fumigatus strain IFM77.

FIG. 19 is an electrophoretogram in the case of using strains ofNeosartorya paulistensis in Example 1(C-4). FIGS. 19 (a) and (b) showelectrophoretograms of samples of strains of Neosartorya paulistensis.FIG. 19(a): Primers N2F/R represented by the nucleotide sequences setforth in SEQ ID NOS: 14 and 15 were used. FIG. 19(b): Primers Af1F/Rrepresented by the nucleotide sequences set forth in SEQ ID NOS: 22 and23 were used. The following strains were used in each of FIGS. 19(a) and(b): lane 1: N. paulistensis CBM-FA-191 81-GU-14-1; lane 2: N.paulistensis CBM-FA-608; and lane 3: N. paulistensis CBM-FA-176. “P” wasthe positive control: A. fumigatus strain IFM77.

FIG. 20 is an electrophoretogram in the case of using strains ofNeosartorya fischeri spinosa in Example 1(C-4). FIGS. 20 (a) and (b)show electrophoretograms of samples of strains of Neosartorya spinosa.FIG. 20(a): Primers N2F/R represented by the nucleotide sequences setforth in SEQ ID NOS: 14 and 15 were used. FIG. 20(b): Primers Af1F/Rrepresented by the nucleotide sequences set forth in SEQ ID NOS: 22 and23 were used. The following strains were used in each of FIGS. 20(a) and(b): lane 1: N. spinosa CBM-FA-892 98-TA-439-N; lane 2: N. spinosaCBM-FA-678 93-BS-620-4; lane 3: N. spinosa XY-9-1; and lane 4: N.spinosa 2000-XY-325-3. “P” was the positive control: A. fumigatus strainIFM77.

FIG. 21 is an electrophoretogram showing discrimination results of fungibelonging to the genus Hamigera in Example 1(D-1).

FIG. 22 is an electrophoretogram showing discrimination results of fungibelonging to the genus Hamigera in Example 1(D-2).

FIG. 23 is an electrophoretogram showing discrimination results of fungibelonging to the genus Hamigera in Example 1(D-3).

FIG. 24-1 is an electrophoretogram in the case of using strains ofHamigera striata in Example 1(D-4).

FIG. 24-2 is an electrophoretogram in the case of using strains ofHamigera avellanea in Example 1(D-5).

FIG. 25-1 is an electrophoretogram in the case of using fungi belongingto the genera Hamigera and Byssochlamys in Example 1(D-6).

FIG. 25-2 is an electrophoretogram in the case of using fungi belongingto the genera Hamigera and Byssochlamys in Example 1(D-6).

FIG. 26 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting the genusByssochlamys in the nucleotide sequences of the ITS region and D1/D2region of 28S rDNA of fungi belonging to the genus the genusByssochlamys (SEQ ID NO:25).

FIG. 27 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting the genusNeosartorya in the nucleotide sequences of the β-tubulin genes of fungibelonging to the genus the genus Neosartorya (SEQ ID NO:88).

FIG. 28 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting Aspergillusfumigatus in the nucleotide sequences of the β-tubulin genes ofAspergillus fumigatus (SEQ ID NO:34).

FIG. 29 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting the genusHamigera in the nucleotide sequences of the β-tubulin genes of fungibelonging to the genus Hamigera (nucleotides 1 to 525 of SEQ ID NO:35are shown).

FIG. 30 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting Talaromycesflavus in the nucleotide sequences of the β-tubulin genes of Talaromycesflavus (SEQ ID NO:26).

FIG. 31 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting Talaromyceswortmannii in the nucleotide sequences of the β-tubulin genes ofTalaromyces wortmannii (SEQ ID NO:29).

FIG. 32 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting Talaromycesluteus in the nucleotide sequences of the β-tubulin genes of Talaromycesluteus (SEQ ID NO:27).

FIG. 33 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting Talaromycesflavus in the nucleotide sequences of the ITS region and D1/D2 region of28S rDNA of Talaromyces flavus (SEQ ID NO:30).

FIG. 34 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting Talaromycestrachyspermus (SEQ ID NO:31) and Talaromyces flavus (SEQ ID NO:30) inthe nucleotide sequences of the ITS region and D1/D2 region of 28S rDNAof Talaromyces trachyspermus and Talaromyces flavus. Nucleotide bases1-567 of SEQ ID NO: 31 and nucleotide bases 1-554 of SEQ ID NO:32 areshown on FIG. 34.

FIG. 34-1 is a diagram illustrating the position relationship ofnucleotide sequences recognized by primers for detecting Talaromycestrachyspermus (SEQ ID NO:31) and Talaromyces flavus (SEQ ID NO:30) inthe nucleotide sequences of the ITS region and D1/D2 region of 28S rDNAof Talaromyces trachyspermus and Talaromyces flavus. (Continuation ofFIG. 34). Nucleotide bases 568-1113 of SEQ ID NO: 31 and nucleotidebases 555-1098 of SEQ ID NO:32 are shown on FIG. 34-1.

FIG. 35 is a graph illustrating the detection sensitivity of the ITSregion and D1/D2 region of 28S rDNA of fungi belonging to the genusByssochlamys by real-time turbidity monitoring method in Example 2. Thenumeral 1 denotes the detection sensitivity of a sample includinggenomic DNA derived from Byssochlamys fulva IFM48421 strain; the numeral2 denotes the detection sensitivity of a sample including genomic DNAderived from Byssochlamys nivea IFM51244 strain, the numeral 4 denotesthe detection sensitivity of a sample including genomic DNA derived fromTalaromyces luteus IFM53241 strain, the numeral 6 denotes the detectionsensitivity of a sample including genomic DNA derived from Talaromyceswortmannii IFM52262 strain, and the numeral 7 denotes the detectionsensitivity of a sample including genomic DNA derived from Neosartoryafischeri IFM46945 strain. FIG. 35(a) shows the results of samples Nos. 1to 8 in Table 15, and FIG. 35(b) shows the results of samples Nos. 9 to16 in Table 15.

FIG. 36 is a graph illustrating the detection sensitivity of theβ-tubulin genes of fungi belonging to the genus Neosartorya by real-timeturbidity monitoring method in Example 3. The numeral 1 denotes thedetection sensitivity of a sample including genomic DNA derived fromNeosartorya fischeri IFM46945 strain; the numeral 2 denotes thedetection sensitivity of a sample including genomic DNA derived fromNeosartorya spinosa IFM46967 strain; the numeral 3 denotes the detectionsensitivity of a sample including genomic DNA derived from Neosartoryaglabra IFM46949 strain; the numeral 4 denotes the detection sensitivityof a sample including genomic DNA derived from Neosartorya hiratsukaeIFM47036 strain; the numeral 5 denotes the detection sensitivity of asample including genomic DNA derived from Talaromyces flavus IFM42243strain; the numeral 6 denotes the detection sensitivity of a sampleincluding genomic DNA derived from Talaromyces luteus IFM53242 strain;the numeral 7 denotes the detection sensitivity of a sample includinggenomic DNA derived from Talaromyces trachyspermus IFM42247 strain; thenumeral 9 denotes the detection sensitivity of a sample includinggenomic DNA derived from Byssochlamys fulva IFM48421 strain; the numeral11 denotes the detection sensitivity of a sample including genomic DNAderived from Alternaria alternate IFM41348 strain; the numeral 14denotes the detection sensitivity of a sample including genomic DNAderived from Fusarium oxysporium IFM50002 strain. FIG. 36(a) shows theresults of samples Nos. 1 to 7 and FIG. 36(b) shows the results ofsamples Nos. 9, 11 and 14.

FIG. 36-1 is a graph illustrating the detection sensitivity of theβ-tubulin genes of fungi belonging to the genus Neosartorya andAspergillus fumigatus by real-time turbidity monitoring method inExample 3.

FIG. 37 is a graph illustrating the detection sensitivity of theβ-tubulin genes of fungi belonging to the genus Hamigera by real-timeturbidity monitoring method in Example 4. The numeral 1 denotes thedetection sensitivity of a sample including genomic DNA derived fromHamigera avellanea IFM42323 strain; the numeral 2 denotes the detectionsensitivity of a sample including genomic DNA derived from Hamigeraavellanea IFM52241 strain; the numeral 3 denotes the detectionsensitivity of a sample including genomic DNA derived from Hamigeraavellanea IFM52957 strain; the numeral 4 denotes the detectionsensitivity of a sample including genomic DNA derived from Byssochlamysfulva IFM51213 strain; the numeral 5 denotes the detection sensitivityof a sample including genomic DNA derived from Byssochlamys niveaIFM51245 strain; the numeral 6 denotes the detection sensitivity of asample including genomic DNA derived from Paecilomyces variotii IFM40913strain; the numeral 7 denotes the detection sensitivity of a sampleincluding genomic DNA derived from Paecilomyces variotii IFM40915strain.

FIG. 38 is a graph illustrating the detection sensitivity of theβ-tubulin genes of Aspergillus fumigatus by real-time turbiditymonitoring method in Example 5. The numeral 1 denotes the detectionsensitivity of a sample including genomic DNA derived from Aspergillusfumigatus A209 strain; the numeral 2 denotes the detection sensitivityof a sample including genomic DNA derived from Aspergillus fumigatusA213 strain; the numeral 3 denotes the detection sensitivity of a sampleincluding genomic DNA derived from Aspergillus fumigatus A215 strain;the numeral 6 denotes the detection sensitivity of a sample includinggenomic DNA derived from Neosartorya spinosa IFM46967 strain.

FIG. 39 is a graph illustrating the detection sensitivity of theβ-tubulin genes of Talaromyces flavus by real-time turbidity monitoringmethod in Example 6. The numeral 1 denotes the detection sensitivity ofa sample including genomic DNA derived from Talaromyces flavus IFM42243strain; the numeral 2 denotes the detection sensitivity of a sampleincluding genomic DNA derived from Talaromyces flavus IFM52233 strain;the numeral 6 denotes the detection sensitivity of a sample includinggenomic DNA derived from Talaromyces trachyspermus IFM52252 strain; thenumeral 7 denotes the detection sensitivity of a sample includinggenomic DNA derived from Talaromyces wortmannii IFM52255 strain; thenumeral 9 denotes the detection sensitivity of a sample includinggenomic DNA derived from Byssochlamys fulva IFM48421 strain. FIG. 39(a)shows the results of samples Nos. 1 to 8 in Table 19, and FIG. 39(b)shows the results of samples Nos. 9 to 16 in Table 19.

FIG. 40 is a graph illustrating the detection sensitivity of theβ-tubulin genes of Talaromyces wortmannii by real-time turbiditymonitoring method in Example 7. The numeral 1 denotes the detectionsensitivity of a sample including genomic DNA derived from Talaromyceswortmannii IFM52255 strain; the numeral 2 denotes the detectionsensitivity of a sample including genomic DNA derived from Talaromyceswortmannii IFM52262 strain; the numeral 3 denotes the detectionsensitivity of a sample including genomic DNA derived from Talaromycesflavus IFM42243 strain; the numeral 4 denotes the detection sensitivityof a sample including genomic DNA derived from Talaromyces luteusIFM53241 strain; the numeral 5 denotes the detection sensitivity of asample including genomic DNA derived from Talaromyces trachyspermusIFM42247 strain; the numeral 7 denotes the detection sensitivity of asample including genomic DNA derived from Byssochlamys nivea IFM51244strain; the numeral 8 denotes the detection sensitivity of a sampleincluding genomic DNA derived from Hamigera avellanea IFM42323 strain.

FIG. 41 is a graph illustrating the detection sensitivity of theβ-tubulin genes of Talaromyces luteus by real-time turbidity monitoringmethod in Example 8. The numeral 1 denotes the detection sensitivity ofa sample including genomic DNA derived from Talaromyces luteus IFM53242strain; the numeral 2 denotes the detection sensitivity of a sampleincluding genomic DNA derived from Talaromyces luteus IFM53241 strain;the numeral 5 denotes the detection sensitivity of a sample includinggenomic DNA derived from Talaromyces wortmannii IFM52262 strain; thenumeral 8 denotes the detection sensitivity of a sample includinggenomic DNA derived from Neosartorya spinosa IFM46967 strain. FIG. 41(a)shows the results of samples Nos. 1 to 8 in Table 21, and FIG. 41(b)shows the results of samples Nos. 9 to 16 in Table 21.

FIG. 42 is a graph illustrating the detection sensitivity of the ITSregion and D1/D2 region of 28S rDNA of Talaromyces flavus andTalaromyces trachyspermus by real-time turbidity monitoring method inExample 9. The numeral 1 denotes the detection sensitivity of a sampleincluding genomic DNA derived from Talaromyces flavus IFM42243 strain;the numeral 2 denotes the detection sensitivity of a sample includinggenomic DNA derived from Talaromyces flavus IFM52233 strain; the numeral3 denotes the detection sensitivity of a sample including genomic DNAderived from Talaromyces flavus T38 strain; the numeral 6 denotes thedetection sensitivity of a sample including genomic DNA derived fromTalaromyces trachyspermus IFM42247 strain; the numeral 7 denotes thedetection sensitivity of a sample including genomic DNA derived fromTalaromyces trachyspermus IFM52252 strain; the numeral 9 denotes thedetection sensitivity of a sample including genomic DNA derived fromTalaromyces wortmannii IFM52255 strain; the numeral 10 denotes thedetection sensitivity of a sample including genomic DNA derived fromByssochlamys fulva IFM48421 strain; the numeral 12 denotes the detectionsensitivity of a sample including genomic DNA derived from Penicilliumgriseofulvum IFM54313 strain; the numeral 13 denotes the detectionsensitivity of a sample including genomic DNA derived from Penicilliumcitirinum IFM54314 strain; the numeral 15 denotes the detectionsensitivity of a sample including genomic DNA derived from Neosartoryaficheri IFM46945 strain; the numeral 16 denotes the detectionsensitivity of a sample utilizing DW as a negative control.

Other and further features and advantages of the invention will appearmore fully from the following description, appropriately referring tothe accompanying drawings.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail.

The present invention relates to a method of specificallydiscriminating/detecting a heat-resistant fungus by identifying theheat-resistant fungus using a nucleic acid including a partialnucleotide sequence of the β-tubulin gene or nucleotide sequences of theD1/D2 region and ITS region of 28S rDNA of the heat-resistant fungus,i.e., a nucleotide sequence of a region specific to each genus of theheat-resistant fungi or a species-specific region (variable region) inthe β-tubulin gene region or the D1/D2 region and ITS region of 28S rDNAof the heat-resistant fungus.

According to the present invention, it is possible todiscriminate/detect a heat-resistant fungus such as a fungus belongingto the genus Byssochlamys, a fungus belonging to the genus Talaromyces,a fungus belonging to the genus Neosartorya, a fungus belonging to thegenus Hamigera, or Aspergillus fumigatus.

More specifically, the present invention is a method of detecting aheat-resistant fungus, including at least one of the followingidentification/detection steps 1) to 4).

1) A step of specifically discriminating/detecting a fungus belonging tothe genus Byssochlamys by identifying the fungus belonging to the genusByssochlamys using a nucleic acid including a nucleotide sequence of aregion specific to the genus Byssochlamys (variable region) in theβ-tubulin gene region and/or the D1/D2 region and ITS region of 28S rDNAof the fungus belonging to the genus Byssochlamys.2) A step of specifically discriminating/detecting a fungus belonging tothe genus Talaromyces by identifying the fungus belonging to the genusTalaromyces using a nucleic acid including a nucleotide sequence of aregion specific to the genus Talaromyces or a species-specific region(variable region) in the β-tubulin gene region and/or the D1/D2 regionand ITS region of 28S rDNA of the fungus belonging to the genusTalaromyces.3) A step of specifically discriminating/detecting a fungus belonging tothe genus Neosartorya and/or Aspergillus fumigatus by identifying thefungus belonging to the genus Neosartorya and/or Aspergillus fumigatususing a nucleic acid including a nucleotide sequence of a regionspecific to the genus Neosartorya and/or Aspergillus fumigatus or aspecies-specific region (variable region) in the β-tubulin gene regionof the fungus belonging to the genus Neosartorya and/or Aspergillusfumigatus.4) A step of specifically discriminating/detecting a fungus belonging tothe genus Hamigera by identifying the fungus belonging to the genusHamigera using a nucleic acid including a nucleotide sequence of aregion specific to the genus Hamigera or a species-specific region(variable region) in the β-tubulin gene region of the fungus belongingto the genus Hamigera.

The detection method of the present invention includes preferably atleast two of the above-mentioned identification/detection steps 1) to4), more preferably at least three of the above-mentionedidentification/detection steps 1) to 4), still more preferably all thesteps of the above-mentioned identification/detection steps 1) to 4). Ifthe detection method of the present invention includes a plurality ofthe above-mentioned steps 1) to 4), it is possible to comprehensivelydetect the heat-resistant fungus which is a main fungi causingcontamination of foods and drinks.

The “heat-resistant fungus” in the present invention, such as the fungusbelonging to the genus Byssochlamys, the fungus belonging to the genusTalaromyces, the fungus belonging to the genus Neosartorya, Aspergillusfumigatus, and the fungus belonging to the genus Hamigera, is aplectomycete belonging to the family Trichocomaceae and is aheat-resistant fungus which forms ascospores which can remain viableeven after a heat treatment at 75° C. for 30 minutes. Examples of thefungus belonging to the genus Byssochlamys include Byssochlamys fulvaand Byssochlamys nivea. Examples of the fungus belonging to the genusTalaromyces include Talaromyces flavus, Talaromyces luteus, Talaromycestrachyspermus, Talaromyces wortmannii, Talaromyces bacillisporus, andTalaromyces macrosporus. Examples of the fungus belonging to the genusNeosartorya include Neosartorya fischeri var. spinosa; (hereinafter,also referred to as “Neosartorya spinosa”), Neosartorya fischeri var.fischeri; (hereinafter, also referred to as “Neosartorya fischeri”),Neosartorya fischeri var. glabra; (hereinafter, also referred to as“Neosartorya glabra”), Neosartorya hiratsukae, Neosartorya paulistensis,and Neosartorya peudofischeri. Examples of the fungus belonging to thegenus Hamigera include Hamigera avellanea, and Hamigera striata.

The “Aspergillus fumigatus” in the present invention is one ofdeuteromycetes and is morphologically very similar to an anamorph(asexual stage) of Neosartorya fischeri but has no teleomorph (sexualstage).

In the present invention, the “variable region” is a region wherenucleotide mutations tend to accumulate in the β-tubulin gene or in theD1/D2 region and ITS (internal transcribed spacer) region of 28S rDNA.

The “β-tubulin” is a protein which constitutes a microtubule withα-tubulin, and the “β-tubulin gene” is a gene encoding β-tubulin. The“28S rDNA” is a DNA encoding gene information of ribosome whereconversion into a protein is performed. The inventors of the presentinvention have focused on that proteins themselves encoded by both theβ-tubulin gene and 28S rDNA are universally present in fungi. Further,the inventors have discovered that nucleotide mutations tend toaccumulate in the β-tubulin gene and 28S rDNA sequence and the mutationsare conserved at genus- or species-level, and there is a highpossibility that a specific region having a nucleotide sequence whichcan be used for discrimination from another genus or species is presentin the β-tubulin gene or 28S rDNA sequence. Based on such findings, theinventors identified/analyzed the nucleotide sequence of the β-tubulingene or 28S rDNA for each of fungi such as the above-mentionedheat-resistant fungi, to thereby determine the “variable regions”according to the present invention.

The nucleotide sequences of the variable regions are significantlydifferent among the genera or species of fungi, and the variable regionsin the β-tubulin genes or the D1/D2 regions and ITS regions of 28S rDNAof the fungi include nucleotide sequences specific to the fungi.Therefore, it is possible to distinguish from other genera or otherspecies of fungi based on the nucleotide sequences of the variableregions.

The nucleotide sequence of the specific region (variable region) in theβ-tubulin gene or the D1/D2 region and ITS region of 28S rDNA of aheat-resistant fungus for use in the present invention corresponds tothe following nucleic acid (I) or (II).

(I) a nucleic acid including a nucleotide sequence set forth in any oneof SEQ ID NOS: 24 to 35 and 83 to 86, or a complementary sequencethereof.

(II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in any one of SEQ ID NOS: 24 to 35 and 83to 86 and being capable of detecting the heat-resistant fungus; or acomplementary sequence thereof.

More specifically, the following nucleic acid (A-I) or (A-II) is anucleic acid corresponding to the nucleotide sequence of the specificregion (variable region) in the β-tubulin gene and/or the D1/D2 regionand ITS region of 28S rDNA of a fungus belonging to the genusByssochlamys, and is used to detect the fungus belonging to the genusByssochlamys.

(A-I) a nucleic acid including a nucleotide sequence set forth in SEQ IDNO: 24 or 25, or a complementary sequence thereof.

(A-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in SEQ ID NO: 24 or 25 and being capableof detecting the fungus belonging to the genus Byssochlamys, or acomplementary sequence thereof.

The inventors of the present invention identified the nucleotidesequences of a variety of the β-tubulin genes and D1/D2 regions and ITSregions of 28S rDNA from fungi belonging to the genus Byssochlamys andrelated species of the fungi belonging to the genus Byssochlamys, andperformed genetic distance analyses between the related genera of thegenus Byssochlamys and the genus Byssochlamys, and among the fungibelonging to the genus Byssochlamys. Moreover, the inventors performedhomology analyses of the determined β-tubulin gene sequences and thenucleotide sequences of the D1/D2 regions and ITS regions of 28S rDNA.As a result, the inventors found out variable regions specific to thefungi belonging to the genus Byssochlamys in the sequences. The variableregion has a specific nucleotide sequence to the fungi belonging to thegenus Byssochlamys, and hence it is possible to discriminate/identifythe fungi belonging to the genus Byssochlamys based on the sequence ofthe variable region.

In the detection method of the present invention, the following nucleicacid (B-I) or (B-II) is a nucleic acid corresponding to the nucleotidesequence of the specific region (variable region) in the β-tubulin geneand/or the D1/D2 region and ITS region of 28S rDNA of a fungus belongingto the genus Talaromyces, and is used to detect the fungus belonging tothe genus Talaromyces.

(B-I) a nucleic acid including a nucleotide sequence set forth in anyone of SEQ ID NOS: 26 to 31, or a complementary sequence thereof.

(B-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in any one of SEQ ID NOS: 26 to 31 andbeing capable of detecting the fungus belonging to the genusTalaromyces, or a complementary sequence thereof.

The inventors of the present invention identified nucleotide sequencesof a variety of the β-tubulin genes and D1/D2 regions and ITS regions of28S rDNA from fungi belonging to the genus Talaromyces and relatedspecies of the fungi belonging to the genus Talaromyces, and performedgenetic distance analyses between the related genera of the genusTalaromyces and the genus Talaromyces, and among the fungi belonging tothe genus Talaromyces. Moreover, the inventors performed homologyanalyses of the determined β-tubulin gene sequences and the nucleotidesequences of the D1/D2 regions and ITS regions of 28S rDNA. As a result,the inventors found out variable regions specific to the fungi belongingto the genus Talaromyces in the sequences. The variable region has aspecific nucleotide sequence to the fungi belonging to the genusTalaromyces, and hence it is possible to discriminate/identify the fungibelonging to the genus Talaromyces based on the sequence of the variableregion.

In the detection method of the present invention, the following nucleicacid (C-I) or (C-II) is a nucleic acid corresponding to the nucleotidesequence of the specific region (variable region) in the β-tubulin geneof a fungus belonging to the genus Neosartorya and/or Aspergillusfumigatus, and is used to detect the fungus belonging to the genusNeosartorya and/or Aspergillus fumigatus.

(C-I) a nucleic acid including a nucleotide sequence set forth in anyone of SEQ ID NOS: 32 to 34 and 83 to 86, or a complementary sequencethereof.

(C-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in any one of SEQ ID NOS: 32 to 34 and 83to 86 and being capable of detecting the fungus belonging to the genusNeosartorya and/or Aspergillus fumigatus, or a complementary sequencethereof.

The inventors of the present invention identified nucleotide sequencesof a variety of the β-tubulin genes from fungi belonging to the genusNeosartorya and/or Aspergillus fumigatus and related species of thefungi belonging to the genus Neosartorya and/or Aspergillus fumigatus,and performed genetic distance analyses between the related genera ofthe genus Neosartorya and the genus Neosartorya, among the fungibelonging to the genus Neosartorya, and among the related genera of thegenus Neosartorya, the genus Neosartorya and Aspergillus fumigatus.Moreover, the inventors performed homology analyses of the determinedβ-tubulin gene sequences. As a result, the inventors found out variableregions specific to the fungi belonging to the genus Neosartorya and/orAspergillus fumigatus in the sequences. The variable regions havespecific nucleotide sequences to the fungi belonging to the genusNeosartorya and Aspergillus fumigatus, and hence it is possible todiscriminate/identify the fungi belonging to the genus Neosartorya andAspergillus fumigatus based on the sequence of the variable region.

In the detection method of the present invention, the following nucleicacid (D-I) or (D-II) is a nucleic acid corresponding to the nucleotidesequence of the specific region (variable region) in the β-tubulin geneof a fungus belonging to the genus Hamigera, and is used to detect thefungus belonging to the genus Hamigera.

(D-I) a nucleic acid including a nucleotide sequence set forth in SEQ IDNO: 35, or a complementary sequence thereof.

(D-II) a nucleic acid including a nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in SEQ ID NO: 35 and being capable ofdetecting the fungus belonging to the genus Hamigera, or a complementarysequence thereof.

The inventors of the present invention identified nucleotide sequencesof a variety of the β-tubulin genes from fungi belonging to the genusHamigera and related species of the fungi belonging to the genusHamigera, and performed genetic distance analyses between the relatedgenera of the genus Hamigera and the genus Hamigera, and among the fungibelonging to the genus Hamigera. Moreover, the inventors performedhomology analyses of the determined β-tubulin gene sequences. As aresult, the inventors found out variable regions specific to the fungibelonging to the genus Hamigera in the sequences. The variable regionhas a specific nucleotide sequence to the fungi belonging to the genusHamigera, and hence it is possible to discriminate/identify the fungibelonging to the genus Hamigera based on the sequence of the variableregion.

In the present invention, such variable regions, and nucleic acids andoligonucleotides derived from such variable regions are used as targets.

The nucleotide sequence (A-I) or (A-II) to be used in the detectionmethod of the present invention corresponds to a partial nucleotidesequence of the β-tubulin gene or the nucleotide sequences of thevariable regions of the ITS region and D1/D2 region of 28S rDNA of thefungi belonging to the genus Byssochlamys.

The nucleotide sequence set forth in SEQ ID NO: 24 or the complementarysequence thereof is the nucleotide sequence of the variable region inthe β-tubulin gene isolated and identified from Byssochlamys nivea. Thenucleotide sequence set forth in SEQ ID NO: 25 or the complementarysequence thereof is the nucleotide sequences of the variable regions inthe ITS region and D1/D2 region of 28S rDNA isolated and identified fromByssochlamys fulva. The sequences are specific to the fungi belonging tothe genus Byssochlamys, and it is possible to specificallydiscriminate/identify the fungi belonging to the genus Byssochlamys byconfirming whether a sample has the nucleotide sequences or not.Moreover, it is also possible to specifically discriminate/identify thefungi belonging to the genus Byssochlamys by using the nucleic acidincluding the nucleotide sequence resulting from a deletion,substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in any one of SEQ ID NOS: 24 to 25 andbeing capable of detecting the fungus belonging to the genusByssochlamys, or the complementary sequence thereof. The nucleic acidsincluding such nucleotide sequences are particularly preferably used fordetecting Byssochlamys nivea and Byssochlamys fulva.

The nucleotide sequence (B-I) or (B-II) to be used in the detectionmethod of the present invention corresponds to a partial nucleotidesequence of the β-tubulin gene or the nucleotide sequences of thevariable regions of the ITS region and D1/D2 region of 28S rDNA of thefungi belonging to the genus Talaromyces.

The nucleotide sequence set forth in SEQ ID NO: 26 or the complementarysequence thereof is the nucleotide sequence of the variable region inthe β-tubulin gene isolated and identified from Talaromyces flavus. Thesequences are specific to the fungi belonging to the genus Talaromyces,and it is possible to specifically discriminate/identify the fungibelonging to the genus Talaromyces by confirming whether a sample hasthe nucleotide sequences or not. Moreover, it is also possible tospecifically discriminate/identify the fungi belonging to the genusTalaromyces by using the nucleic acid including the nucleotide sequenceresulting from a deletion, substitution, or addition of one to severalnucleotides in the nucleotide sequence set forth in SEQ ID NO: 26 andbeing capable of detecting the fungi belonging to the genus Talaromyces,or the complementary sequence thereof. The nucleic acids including suchnucleotide sequences are particularly preferably used for detectingTalaromyces flavus and Talaromyces trachyspermus.

The nucleotide sequence set forth in SEQ ID NO: 27 or the complementarysequence thereof is the nucleotide sequence of the variable region inthe β-tubulin gene isolated and identified from Talaromyces luteus. Thesequences are specific to the fungi belonging to the genus Talaromyces,and it is possible to specifically discriminate/identify the fungibelonging to the genus Talaromyces by confirming whether a sample hasthe nucleotide sequences or not. Moreover, it is also possible tospecifically discriminate/identify the fungi belonging to the genusTalaromyces by using the nucleic acid including the nucleotide sequenceresulting from a deletion, substitution, or addition of one to severalnucleotides in the nucleotide sequence set forth in SEQ ID NO: 27 andbeing capable of detecting the fungi belonging to the genus Talaromyces,or the complementary sequence thereof. The nucleic acids including suchnucleotide sequences are particularly preferably used for detectingTalaromyces luteus, Talaromyces bacillisporus and Talaromyceswortmannii.

The nucleotide sequence set forth in SEQ ID NO: 28 or the complementarysequence thereof is the nucleotide sequences of the variable regions inthe ITS region and D1/D2 region of 28S rDNA isolated and identified fromTalaromyces wortmannii. The sequences are specific to the fungibelonging to the genus Talaromyces, and it is possible to specificallydiscriminate/identify the fungi belonging to the genus Talaromyces byconfirming whether a sample has the nucleotide sequences or not.Moreover, it is also possible to specifically discriminate/identify thefungi belonging to the genus Talaromyces by using the nucleic acidincluding the nucleotide sequence resulting from a deletion,substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in SEQ ID NO: 28 and being capable ofdetecting the fungi belonging to the genus Talaromyces, or thecomplementary sequence thereof. The nucleic acids including suchnucleotide sequences are particularly preferably used for detectingTalaromyces wortmannii, Talaromyces flavus, Talaromyces trachyspermusand Talaromyces macrosporus.

The nucleotide sequence set forth in SEQ ID NO: 29 or the complementarysequence thereof is the nucleotide sequence of the variable region inthe β-tubulin gene isolated and identified from Talaromyces wortmannii.The sequences are specific to the fungi belonging to the genusTalaromyces, and it is possible to specifically discriminate/identifythe fungi belonging to the genus Talaromyces by confirming whether asample has the nucleotide sequences or not. Moreover, it is alsopossible to specifically discriminate/identify the fungi belonging tothe genus Talaromyces by using the nucleic acid including the nucleotidesequence resulting from a deletion, substitution, or addition of one toseveral nucleotides in the nucleotide sequence set forth in SEQ ID NO:29 and being capable of detecting the fungi belonging to the genusTalaromyces, or the complementary sequence thereof. The nucleic acidsincluding such nucleotide sequences are particularly preferably used fordetecting Talaromyces wortmannii.

The nucleotide sequence set forth in SEQ ID NO: 30 or the complementarysequence thereof is the nucleotide sequences of the variable regions inthe ITS region and D1/D2 region of 28S rDNA isolated and identified fromTalaromyces flavus. The nucleotide sequence set forth in SEQ ID NO: 31or the complementary sequence thereof is the nucleotide sequences of thevariable regions in the ITS region and D1/D2 region of 28S rDNA isolatedand identified from Talaromyces trachyspermus. The sequences arespecific to the fungi belonging to the genus Talaromyces, and it ispossible to specifically discriminate/identify the fungi belonging tothe genus Talaromyces by confirming whether a sample has the nucleotidesequences or not. Moreover, it is also possible to specificallydiscriminate/identify the fungi belonging to the genus Talaromyces byusing the nucleic acid including the nucleotide sequence resulting froma deletion, substitution, or addition of one to several nucleotides inthe nucleotide sequence set forth in SEQ ID NO: 30 or 31 and beingcapable of detecting the fungi belonging to the genus Talaromyces, orthe complementary sequence thereof. The nucleic acids including suchnucleotide sequences are particularly preferably used for detectingTalaromyces flavus and Talaromyces trachyspermus.

The nucleotide sequence (C-I) or (C-II) to be used in the detectionmethod of the present invention corresponds to a partial nucleotidesequence of the β-tubulin gene of the fungi belonging to the genusNeosartorya or Aspergillus fumigatus.

The nucleotide sequence set forth in SEQ ID NO: 32 or the complementarysequence thereof is the nucleotide sequence of the variable region inthe β-tubulin gene isolated and identified from Neosartorya glabra. Thenucleotide sequence set forth in SEQ ID NO: 83 or 84 or thecomplementary sequence thereof is the nucleotide sequence of thevariable region in the β-tubulin gene isolated and identified fromNeosartorya fischeri. The nucleotide sequence set forth in SEQ ID NO: 85or 86 or the complementary sequence thereof is the nucleotide sequenceof the variable region in the β-tubulin gene isolated and identifiedfrom Neosartorya spinosa. The nucleotide sequence set forth in SEQ IDNO: 33 or 34 or the complementary sequence thereof is the nucleotidesequence of the variable region in the β-tubulin gene isolated andidentified from Aspergillus fumigatus. The sequences are specific to thefungi belonging to the genus Neosartorya and/or Aspergillus fumigatus,and it is possible to specifically discriminate/identify the fungibelonging to the genus Neosartorya and/or Aspergillus fumigatus byconfirming whether a sample has the nucleotide sequences or not.Moreover, it is also possible to specifically discriminate/identify thefungi belonging to the genus Neosartorya and/or Aspergillus fumigatus byusing the nucleic acid including the nucleotide sequence resulting froma deletion, substitution, or addition of one to several nucleotides inthe nucleotide sequence set forth in any one of SEQ ID NOS: 32 to 34 and83 to 86 and being capable of detecting the fungi belonging to the genusNeosartorya and/or Aspergillus fumigatus, or the complementary sequencethereof. The nucleic acids including such nucleotide sequences areparticularly preferably used for detecting Neosartorya glabra,Neosartorya fischeri, Neosartorya spinosa, Neosartorya hiratsukae,Neosartorya paulistensis, Neosartorya pseudofischeri, and Aspergillusfumigatus.

The nucleotide sequence (D-I) or (D-II) to be used in the detectionmethod of the present invention corresponds to a partial nucleotidesequence of the β-tubulin gene of the fungi belonging to the genusHamigera.

The nucleotide sequence set forth in SEQ ID NO: 35 or the complementarysequence thereof is the nucleotide sequence of the variable region inthe β-tubulin gene isolated and identified from Hamigera avellanea. Thesequences are specific to the fungi belonging to the genus Hamigera, andit is possible to specifically discriminate/identify the fungi belongingto the genus Hamigera by confirming whether a sample has the nucleotidesequences or not. Moreover, it is also possible to specificallydiscriminate/identify the fungi belonging to the genus Hamigera by usingthe nucleic acid including the nucleotide sequence resulting from adeletion, substitution, or addition of one to several nucleotides in thenucleotide sequence set forth in SEQ ID NO: 35 and being capable ofdetecting the fungi belonging to the genus Hamigera, or thecomplementary sequence thereof. The nucleic acids including suchnucleotide sequences are particularly preferably used for detectingHamigera avellanea and Hamigera striata.

Hereinafter, any one of the above nucleotide sequences (A-I) to (D-II)are also referred to as “the nucleotide sequence of the variable regionaccording to the present invention”

In the present invention, the method of identifying the heat-resistantfungus by using the nucleic acid including the nucleotide sequence ofthe variable region according to the present invention is notparticularly limited, and may be performed by a usual geneticengineering procedure such as a sequencing method, a hybridizationmethod, a PCR method, or a LAMP method.

In the detection method of the present invention for identifying theheat-resistant fungus by using the nucleic acid including the nucleotidesequence of the variable region according to the present invention, apreferable embodiment includes determining a nucleotide sequence of theβ-tubulin gene region or the ITS region and D1/D2 region of 28S rDNA ina sample, and then confirming whether the obtained nucleotide sequenceincludes any one of the nucleotide sequences (A-I) to (D-II) or not.

When identifying the fungi belonging to the genus Byssochlamys by usingthe nucleic acid including the nucleotide sequence of the variableregion, it is preferable to determine a nucleotide sequence of theβ-tubulin gene region or the ITS region and D1/D2 region of 28S rDNA ina sample, and then to confirm whether the obtained nucleotide sequenceincludes the nucleotide sequence (A-I) or (A-II) or not. In other words,the detection method of the present invention preferably includes:analyzing and determining a nucleotide sequence of the β-tubulin gene orthe ITS region and D1/D2 region of 28S rDNA in a sample; comparing thedetermined nucleotide sequence with the nucleotide sequence (A-1) or(A-II) corresponding to the variable region in the β-tubulin gene or theITS region and D1/D2 region of 28S rDNA; and identifying the fungibelonging to the genus Byssochlamys based on the matching or differencebetween the both nucleotide sequences.

When identifying the fungi belonging to the genus Talaromyces by usingthe nucleic acid including the nucleotide sequence of the variableregion, it is preferable to determine a nucleotide sequence of theβ-tubulin gene region or the ITS region and D1/D2 region of 28S rDNA ina sample, and then to confirm whether the obtained nucleotide sequenceincludes the nucleotide sequence (B-I) or (B-II) or not. In other words,the detection method of the present invention preferably includes:analyzing and determining a nucleotide sequence of the β-tubulin gene orthe ITS region and D1/D2 region of 28S rDNA in a sample; comparing thedetermined nucleotide sequence with the nucleotide sequence (B-I) or(B-II) corresponding to the variable region in the β-tubulin gene or theITS region and D1/D2 region of 28S rDNA; and identifying the fungibelonging to the genus Talaromyces based on the matching or differencebetween the both nucleotide sequences.

When identifying the fungi belonging to the genus Neosartorya and/orAspergillus fumigatus by using the nucleic acid including the nucleotidesequence of the variable region, it is preferable to determine anucleotide sequence of the β-tubulin gene region in a sample, and thento confirm whether the obtained nucleotide sequence includes thenucleotide sequence (C-I) or (C-II) or not. In other words, thedetection method of the present invention preferably includes: analyzingand determining a nucleotide sequence of the β-tubulin gene in a sample;comparing the determined nucleotide sequence with the nucleotidesequence (C-I) or (C-II) corresponding to the variable region in theβ-tubulin gene; and identifying the fungi belonging to the genusNeosartorya and/or Aspergillus fumigatus based on the matching ordifference between the both nucleotide sequences.

When identifying the fungi belonging to the genus Hamigera by using thenucleic acid including the nucleotide sequence of the variable region,it is preferable to determine a nucleotide sequence of the β-tubulingene region in a sample, and then to confirm whether the obtainednucleotide sequence includes the nucleotide sequence (D-I) or (D-II) ornot. In other words, the detection method of the present inventionpreferably includes: analyzing and determining a nucleotide sequence ofthe β-tubulin gene in a sample; comparing the determined nucleotidesequence with the nucleotide sequence (D-I) or (D-II) corresponding tothe variable region in the β-tubulin gene; and identifying the fungibelonging to the genus Hamigera based on the matching or differencebetween the both nucleotide sequences.

The method of analyzing and determining the nucleotide sequence is notparticularly limited, and usual RNA or DNA sequencing means may be used.

Specific examples of the method include an electrophoresis method suchas a Maxam-Gilbert method or a Sanger method, mass spectrometry, and ahybridization method. Examples of the Sanger method include a method oflabeling a primer or terminator by a radiation labeling method, afluorescent labeling method, or the like.

In the method of the present invention for detecting/identifying theheat-resistant fungus by using the nucleic acid including the nucleotidesequence of the variable region according to the present invention, anoligonucleotide for detection can be used. The oligonucleotide iscapable of hybridizing with the nucleotide sequence of the variableregion (i.e., any one of the nucleic acids (A-I) to (D-II)), and has afunction as an oligonucleotide for specifically detecting theheat-resistant fungus.

When identifying the fungi belonging to the genus Byssochlamys, anoligonucleotide which is capable of hybridizing with the nucleotidesequence of the variable region (i.e., the nucleic acids (A-I) or(A-II)), and has a function as an oligonucleotide for specificallydetecting the fungi belonging to the genus Byssochlamys, can be used.

When identifying the fungi belonging to the genus Talaromyces, anoligonucleotide which is capable of hybridizing with the nucleotidesequence of the variable region (i.e., the nucleic acids (B-I) or(B-II)), and has a function as an oligonucleotide for specificallydetecting the fungi belonging to the genus Talaromyces, can be used.

When identifying the fungi belonging to the genus Neosartorya and/orAspergillus fumigatus, an oligonucleotide which is capable ofhybridizing with the nucleotide sequence of the variable region (i.e.,the nucleic acids (C-I) or (C-II)), and has a function as anoligonucleotide for specifically detecting the fungi belonging to thegenus Neosartorya and/or Aspergillus fumigatus, can be used.

When identifying the fungi belonging to the genus Hamigera, anoligonucleotide which is capable of hybridizing with the nucleotidesequence of the variable region (i.e., the nucleic acids (D-I) or(D-H)), and has a function as an oligonucleotide for specificallydetecting the fungi belonging to the genus Hamigera, can be used.

The oligonucleotide for detection of the present invention may be onewhich is capable of detecting the heat-resistant fungus. That is, theoligonucleotide may be one which can be used as a nucleic acid primer ora nucleic acid probe for detection of the heat-resistant fungus, or onewhich is capable of hybridizing with the nucleotide sequence of thevariable region in the β-tubulin gene or the ITS region and D1/D2 regionof 28S rDNA of the heat-resistant fungus under stringent conditions. Itshould be note that, in this description, the “stringent conditions”includes, for example, the method described in Molecular Cloning—ALABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell. ColdSpring Harbor Laboratory Press], and examples thereof include conditionswhere hybridization is performed by incubating a solution containing6×SSC (composition of 1×SSC: 0.15M sodium chloride, 0.015M sodiumcitrate, pH7.0), 0.5% SDS, 5×Denhardt and 100 mg/mL herring sperm DNAtogether with a probe at 65° C. for 8 to 16 hours.

The oligonucleotide for detection of the present invention is preferablyan oligonucleotide which is capable of hybridizing with a regionselected from the nucleotide sequences of the variable regions of theβ-tubulin gene or the ITS region and D1/D2 region of 28S rDNA (i.e., aregion in the nucleic acid (I) or (II)), and satisfies the followingfour conditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to each genus of the heat-resistant fungi in thenucleic acid (I) or (II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value (melting temperature) of about55° C. to 65° C.

Specifically, the oligonucleotide for detecting the fungus belonging tothe genus Byssochlamys is preferably an oligonucleotide which is capableof hybridizing with a region in the nucleic acid (A-I) or (A-II) andsatisfies the following four conditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Byssochlamys inthe nucleic acid (A-I) or (A-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value (melting temperature) of about55° C. to 65° C.

The oligonucleotide for detecting the fungus belonging to the genusTalaromyces is preferably an oligonucleotide which is capable ofhybridizing with a region in the nucleic acid (B-I) or (B-II) andsatisfies the following four conditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Talaromyces inthe nucleic acid (B-I) or (B-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value (melting temperature) of about55° C. to 65° C.

The oligonucleotide for detecting the fungus belonging to the genusNeosartorya and/or Aspergillus fumigatus is preferably anoligonucleotide which is capable of hybridizing with a region in thenucleic acid (C-I) or (C-II) and satisfies the following fourconditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Neosartoryaand/or Aspergillus fumigatus in the nucleic acid (C-I) or (C-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value (melting temperature) of about55° C. to 65° C.

The oligonucleotide for detecting the fungus belonging to the genusHamigera is preferably an oligonucleotide which is capable ofhybridizing with a region in the nucleic acid (D-I) or (D-II) andsatisfies the following four conditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Hamigera in thenucleic acid (D-I) or (D-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value (melting temperature) of about55° C. to 65° C.

In the (1) above, the “region containing about 10 continuous nucleotidesspecific to each genus of heat-resistant fungi in the nucleic acid (I)or (II)” refers to a region where the nucleotide sequences betweendifferent genera of the heat-resistant fungi are particularly poorlyconserved (that is, the region has particularly high specificity to eachgenus of the heat-resistant fungi) in the nucleotide sequences of thevariable regions of the β-tubulin gene or the ITS region and D1/D2region of 28S rDNA, and where a nucleotide sequence including about 10continuous nucleotides specific to each genus of the heat-resistantfungi is present.

Specifically, “the region containing about 10 continuous nucleotidesspecific to the fungus belonging to the genus Byssochlamys in thenucleic acid (A-I) or (A-II)” refers to a region where the nucleotidesequences of different fungi are particularly poorly conserved (that is,the region has particularly high specificity to the genus Byssochlamys)in the variable regions of the β-tubulin gene or the ITS region andD1/D2 region of 28S rDNA of the present invention and where a nucleotidesequence including about 10 continuous nucleotides specific to the genusByssochlamys is present.

“The region containing about 10 continuous nucleotides specific to thefungus belonging to the genus Talaromyces in the nucleic acid (B-I) or(B-II)” refers to a region where the nucleotide sequences of differentfungi are particularly poorly conserved (that is, the region hasparticularly high specificity to the genus Talaromyces) in the variableregions of the β-tubulin gene or the ITS region and D1/D2 region of 28SrDNA of the present invention and where a nucleotide sequence includingabout 10 continuous nucleotides specific to the genus Talaromyces ispresent.

“The region containing about 10 continuous nucleotides specific to thefungus belonging to the genus Neosartorya and/or Aspergillus fumigatusin the nucleic acid (C-I) or (C-II)” refers to a region where thenucleotide sequences of different fungi are particularly poorlyconserved (that is, the region has particularly high specificity to thegenus Neosartorya and/or Aspergillus fumigatus) in the variable regionsof the β-tubulin gene of the present invention and where a nucleotidesequence including about 10 continuous nucleotides specific to the genusNeosartorya and/or Aspergillus fumigatus is present.

“The region containing about 10 continuous nucleotides specific to thefungus belonging to the genus Hamigera in the nucleic acid (D-I) or(D-II)” refers to a region where the nucleotide sequences of differentfungi are particularly poorly conserved (that is, the region hasparticularly high specificity to the genus Hamigera) in the variableregions of the β-tubulin gene of the present invention and where anucleotide sequence including about 10 continuous nucleotides specificto the genus Hamigera is present.

Moreover, in the (3) above, the “oligonucleotide has low possibility tocause self-annealing” means that the primers are expected not to bind toeach other from the nucleotide sequences of the primers.

The number of nucleotides in the oligonucleotide for detection of thepresent invention is not particularly limited, and is preferably 13 to30, more preferably 18 to 23. The Tm value of the oligonucleotide inhybridization is preferably in a range of 55° C. to 65° C., morepreferably 59° C. to 62° C. The GC content in the oligonucleotide ispreferably 30% to 80%, more preferably 45% to 65%, most preferably about55%.

The oligonucleotide for detection of the present invention is preferablyan oligonucleotide including the nucleotide sequence set forth in anyone of SEQ ID NOS: 1 to 23 and 36 to 78, or the complementary sequencethereof, or an oligonucleotide including a nucleotide sequence which has70% or more homology to the nucleotide sequence set forth in any one ofSEQ ID NOS: 1 to 23 and 36 to 78, or the complementary sequence thereof,and is capable of detecting the heat-resistant fungi (an oligonucleotidewhich has a function as an oligonucleotide for detection). Theoligonucleotide which is capable of detecting the heat-resistant fungimay be one including a nucleotide sequence which has 70% or morehomology to the nucleotide sequence set forth in any one of SEQ ID NOS:1 to 23 and 36 to 78 and has a function as a nucleic acid primer ornucleic acid probe for detecting the heat-resistant fungi, or mayinclude a nucleotide sequence which can hybridize with the β-tubulingene or the ITS region and D1/D2 region of 28S rDNA of each of theheat-resistant fungi under stringent conditions.

It should be note that, in this description, the “stringent conditions”includes, for example, the method described in Molecular Cloning—ALABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell. ColdSpring Harbor Laboratory Press], and examples thereof include conditionswhere hybridization is performed by incubating a solution containing6×SSC (composition of 1×SSC: 0.15M sodium chloride, 0.015M sodiumcitrate, pH7.0), 0.5% SDS, 5×Denhardt and 100 mg/mL herring sperm DNAtogether with a probe at 65° C. for 8 to 16 hours.

In such oligonucleotide of the present invention, the homology is morepreferably 75% or more, still more preferably 80% or more, even morepreferably 85% or more, further more preferably 90% or more, especiallypreferably 95% or more as long as the oligonucleotide is capable ofdetecting the above-mentioned heat-resistant fungi.

The nucleotide sequence homology is calculated, for example, byLipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it canbe calculated by performing analysis using a homology analysis (Searchhomology) program of genetic information processing software Genetyx-Win(Software Development) while the unit size to compare (ktup) parameteris set to 2.

Further, the oligonucleotide for detection of the present inventionincludes an oligonucleotide obtained by performing a mutation ormodification such as a deletion, insertion, or substitution ofnucleotide(s) for the oligonucleotide including the nucleotide sequenceset forth in any one of SEQ ID NOS: 1 to 23 and 36 to 78 as long as theoligonucleotide is capable of detecting the heat-resistant fungi. Theoligonucleotide of the present invention may be including a nucleotidesequence one which obtained by performing a mutation or modificationsuch as a deletion, insertion, or substitution of nucleotide(s) for theoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 1 to 23 and 36 to 78 and has a function as a nucleic acidprimer or nucleic acid probe for detecting the heat-resistant fungi, ormay include a nucleotide sequence which can hybridize with the β-tubulingene or the ITS region and D1/D2 region of 28S rDNA of each of theheat-resistant fungi under stringent conditions.

It should be note that, in this description, the “stringent conditions”includes, for example, the method described in Molecular Cloning—ALABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell. ColdSpring Harbor Laboratory Press], and examples thereof include conditionswhere hybridization is performed by incubating a solution containing6×SSC (composition of 1×SSC: 0.15M sodium chloride, 0.015M sodiumcitrate, pH7.0), 0.5% SDS, 5×Denhardt and 100 mg/mL herring sperm DNAtogether with a probe at 65° C. for 8 to 16 hours.

The oligonucleotide obtained by performing a mutation or modificationsuch as a deletion, insertion, or substitution of nucleotide(s) includesan oligonucleotide including a nucleotide sequence modified by amutation or modification such as a deletion, insertion or substitutionof one to several, preferably one to five, more preferably one to four,still more preferably one to three, even more preferably one to two,particularly preferably one nucleotide, to the nucleotide sequences setforth in any one of SEQ ID NOS: 1 to 23 and 36 to 78 or thecomplementary sequence thereof. Moreover, an appropriate nucleotidesequence may be added to the nucleotide sequence set forth in any one ofSEQ ID NOS: 1 to 23 and 36 to 78 or the complementary sequence thereof.

In the present invention, among the above-mentioned oligonucleotides fordetection, it is preferable to use an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 1 to 23 and 36to 78, or an oligonucleotide including the nucleotide sequence which has70% or more homology to the nucleotide sequence and has a function as anoligonucleotide for detection; and more preferable to use anoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 1 to 23 and 36 to 78.

More specifically, when identifying/detecting the fungi belonging to thegenus Byssochlamys of the heat-resistant fungi, it is preferable to usean oligonucleotide including the nucleotide sequence set forth in anyone of SEQ ID NOS: 1, 2 and 36 to 39, or the complementary sequencethereof; or an oligonucleotide including the nucleotide sequence whichhas 70% or more homology to the nucleotide sequence set forth in any oneof SEQ ID NOS: 1, 2 and 36 to 39 or the complementary sequence thereof,and which has a function as an oligonucleotide for detection; morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 1, 2 and 36 to 39; or an oligonucleotideincluding the nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection; and still more preferably an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 1, 2 and 36 to39.

When identifying/detecting the fungi belonging to the genus Talaromyces,it is preferable to use an oligonucleotide including the nucleotidesequence set forth in any one of SEQ ID NOS: 3 to 11 and 57 to 78, orthe complementary sequence thereof; or an oligonucleotide including thenucleotide sequence which has 70% or more homology to the nucleotidesequence set forth in any one of SEQ ID NOS: 3 to 11 and 57 to 78 or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection; more preferably an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 3to 11 and 57 to 78; or an oligonucleotide including the nucleotidesequence which has 70% or more homology to the nucleotide sequence andwhich has a function as an oligonucleotide for detection; and still morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 3 to 11 and 57 to 78.

When identifying/detecting the fungi belonging to the genus Neosartoryaand/or Aspergillus fumigatus, it is preferable to use an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 12to 15, 22, 23 and 40 to 50, or the complementary sequence thereof; or anoligonucleotide including the nucleotide sequence which has 70% or morehomology to the nucleotide sequence set forth in any one of SEQ ID NOS:12 to 15, 22, 23 and 40 to 50 or the complementary sequence thereof, andwhich has a function as an oligonucleotide for detection; morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 12 to 15, 22, 23 and 40 to 50, or anoligonucleotide including the nucleotide sequence which has 70% or morehomology to the nucleotide sequence and which has a function as anoligonucleotide for detection; and still more preferably anoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 12 to 15, 22, 23 and 40 to 50.

When identifying/detecting the fungi belonging to the genus Hamigera, itis preferable to use an oligonucleotide including the nucleotidesequence set forth in any one of SEQ ID NOS: 16 to 21 and 51 to 56, orthe complementary sequence thereof; or an oligonucleotide including thenucleotide sequence which has 70% or more homology to the nucleotidesequence set forth in any one of SEQ ID NOS: 16 to 21 and 51 to 56 orthe complementary sequence thereof, and which has a function as anoligonucleotide for detection; more preferably an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 16to 21 and 51 to 56; or an oligonucleotide including the nucleotidesequence which has 70% or more homology to the nucleotide sequence andwhich has a function as an oligonucleotide for detection; and still morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 16 to 21 and 51 to 56.

The above oligonucleotide for detection of the present invention can bepreferably used as a nucleic acid primer and a nucleic acid probe, asdescribed later.

The bonding pattern of the oligonucleotide for detection includes notonly a phosphodiester bond in a natural nucleic acid, but also aphosphoroamidate bond, a phosphorothioate bond and the like.

The oligonucleotide for use in the present invention can be synthesizedby known methods. For example, the oligonucleotide may be chemicallysynthesized based on designed sequences, or purchased from amanufacturer of reagents. Specifically, the oligonucleotide may besynthesized using an oligonucleotide synthesizer or the like. Moreover,after synthesis, the oligonucleotides may be purified by an adsorptioncolumn, high-performance liquid chromatography, or electrophoresis.Furthermore, an oligonucleotide having a nucleotide sequence with asubstitution, deletion, insertion, or addition of one to severalnucleotides may be synthesized by known methods.

In the method of the present invention for identifying theheat-resistant fungus by using the nucleic acid including the nucleotidesequence of the variable region according to the present invention, apreferable embodiment includes labeling an oligonucleotide for detectionwhich is capable of hybridizing with any one of the nucleic acids (A-I)to (D-II) under stringent conditions; hybridizing the resultantoligonucleotide for detection with nucleic acid extracted from a testobject under a stringent condition; and measuring the label of thehybridized oligonucleotide for detection.

In this case, the above-mentioned oligonucleotides for detection of thepresent invention can be used as the oligonucleotide for detection whichis capable of hybridizing with any one of the nucleic acids (A-I) to(D-II) under stringent conditions, and preferred ranges are the same asabove. Among these, it is preferable to use an oligonucleotide includingthe nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 23 and36 to 78, or the complementary sequence thereof; or an oligonucleotideincluding the nucleotide sequence which has 70% or more homology to thenucleotide sequence set forth in any one of SEQ ID NOS: 1 to 23 and 36to 78, or the complementary sequence thereof, and which has a functionas an oligonucleotide for detection; more preferable to use anoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 1 to 23 and 36 to 78, or an oligonucleotide including thenucleotide sequence which has 70% or more homology to the nucleotidesequence and which has a function as an oligonucleotide for detection;and still more preferable to use an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 1 to 23 and 36to 78.

More specifically, when identifying/detecting the fungi belonging to thegenus Byssochlamys of the heat-resistant fungi, it is preferable to usean oligonucleotide including the nucleotide sequence set forth in anyone of SEQ ID NOS: 1, 2 and 36 to 39, or the complementary sequencethereof; or an oligonucleotide including the nucleotide sequence whichhas 70% or more homology to the nucleotide sequence set forth in any oneof SEQ ID NOS: 1, 2 and 36 to 39 or the complementary sequence thereof,and which has a function as an oligonucleotide for detection; morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 1, 2 and 36 to 39, or an oligonucleotideincluding the nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection; and still more preferably an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 1, 2 and 36 to39.

When identifying/detecting the fungi belonging to the genus Talaromyces,it is preferable to use an oligonucleotide including the nucleotidesequence set forth in any one of SEQ ID NOS: 3 to 11 and 57 to 78, orthe complementary sequence thereof; or an oligonucleotide including thenucleotide sequence which has 70% or more homology to the nucleotidesequence set forth in any one of SEQ ID NOS: 3 to 11 and 57 to 78 or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection; more preferably an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 3to 11 and 57 to 78, or an oligonucleotide including the nucleotidesequence which has 70% or more homology to the nucleotide sequence andwhich has a function as an oligonucleotide for detection; and still morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 3 to 11 and 57 to 78.

When identifying/detecting the fungi belonging to the genus Neosartoryaand/or Aspergillus fumigatus, it is preferable to use an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 12to 15, 22, 23 and 40 to 50, or the complementary sequence thereof; or anoligonucleotide including the nucleotide sequence which has 70% or morehomology to the nucleotide sequence set forth in any one of SEQ ID NOS:12 to 15, 22, 23 and 40 to 50 or the complementary sequence thereof, andwhich has a function as an oligonucleotide for detection; morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 12 to 15, 22, 23 and 40 to 50, or anoligonucleotide including the nucleotide sequence which has 70% or morehomology to the nucleotide sequence and which has a function as anoligonucleotide for detection; and still more preferably anoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 12 to 15, 22, 23 and 40 to 50.

When identifying/detecting the fungi belonging to the genus Hamigera, itis preferable to use an oligonucleotide including the nucleotidesequence set forth in any one of SEQ ID NOS: 16 to 21 and 51 to 56, orthe complementary sequence thereof; or an oligonucleotide including thenucleotide sequence which has 70% or more homology to the nucleotidesequence set forth in any one of SEQ ID NOS: 16 to 21 and 51 to 56 orthe complementary sequence thereof, and which has a function as anoligonucleotide for detection; more preferably an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 16to 21 and 51 to 56; or an oligonucleotide including the nucleotidesequence which has 70% or more homology to the nucleotide sequence andwhich has a function as an oligonucleotide for detection; and still morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 16 to 21 and 51 to 56.

The oligonucleotide for detection of the present invention can be usedas a nucleic acid probe. The nucleic acid probe can be prepared bylabeling the above-mentioned oligonucleotide with a labeling substance.The labeling substance is not particularly limited and may include ausual labeling substance such as a radioactive substance, an enzyme, afluorescent substance, a luminescent substance, an antigen, a hapten, anenzyme substrate, or an insoluble carrier. The oligonucleotide may belabeled at its terminal or at the sequence other than the terminals, orat the sugar, phosphate group, or base moiety. Since the nucleic acidprobe can be hybridized specifically with part of the variable region inthe β-tubulin gene or the ITS region and D1/D2 region of 28S rDNA of theheat-resistant fungi, it is possible to rapidly and easily detect theheat-resistant fungi in a sample. Examples of means for detecting thelabel include: autoradiography in the case of a nucleic acid probelabeled with a radioisotope; a fluorescent microscope in the case of anucleic acid probe labeled with a fluorescent substance; and an analysisusing a sensitive film or a digital analysis using a CCD camera in thecase of a nucleic acid probe labeled with a chemiluminescent substance.

The heat-resistant fungi can be detected by hybridizing the thus-labeledoligonucleotide for detection of the present invention with a nucleicacid extracted from a test object by a usual method under stringentconditions; and measuring the label of the hybridized oligonucleotidefor detection. It this case, the stringent conditions may be the sameconditions as described above. As a method of measuring the label of thenucleic acid probe hybridized with nucleic acid, a usual method (such asa FISH method, a dot-blot method, a Southern-blot method, or aNorthern-blot method) may be used.

Further, the oligonucleotide for use in the present invention may bebound to a solid-phase carrier and used as a capture probe. In thiscase, the capture probe and labeled nucleic acid probe may be combinedand used in a sandwich assay, or a target nucleic acid may be labeledand captured.

An example of the detection method using the oligonucleotide of thepresent invention as nucleic acid probe is shown below.

In the case of detection of the fungus belonging to the genusNeosartorya and/or Aspergillus fumigatus, the following oligonucleotides(I) to (w) may be labeled to prepare nucleic acid probes. As mentionedlater, the nucleic acid probes consisting of the followingoligonucleotides (l) to (o) hybridize specifically with parts of thevariable regions of the β-tubulin genes of the fungus belonging to thegenus Neosartorya and Aspergillus fumigatus, and hence can rapidly andeasily detect the fungi belonging to the genus Neosartorya andAspergillus fumigatus in samples. The nucleic acid probe consisting ofthe following oligonucleotides (v) and/or (w) hybridizes specificallywith part of the variable region in the β-tubulin gene of Aspergillusfumigatus but cannot hybridize with DNA and RNA of the fungus belongingto the genus Neosartorya. Therefore, it is possible to rapidly andeasily discriminate the fungus in the sample as one belonging to thegenus Neosartorya or Aspergillus fumigatus by confirming whether theoligonucleotides hybridize with the region or not.

In the method of the present invention for identifying theheat-resistant fungus by using the nucleic acid including the nucleotidesequence of the variable region according to the present invention, apreferable embodiment includes performing gene amplification of anucleic acid consisting of the whole or part of the region of any one ofthe above nucleic acids (A-I) to (D-II), and confirming whether theamplification product is present or not. In this case, theabove-mentioned oligonucleotides for detection of the present inventioncan be used as nucleic acid primers and a pair of nucleic acid primers,and preferred ranges are the same as above. Among theseoligonucleotides, as the nucleic acid primers, it is preferable to usean oligonucleotide which is capable of hybridizing with a region in theabove nucleic acid (I) or (II), and satisfies the following fourconditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to each genus of the heat-resistant fungi in thenucleic acid (I) or (II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value (melting temperature) of about55° C. to 65° C.

Further, as the nucleic acid primers, it is preferable to use anoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 1 to 23 and 36 to 78, or the complementary sequencethereof; or an oligonucleotide including the nucleotide sequence whichhas 70% or more homology to the nucleotide sequence set forth in any oneof SEQ ID NOS: 1 to 23 and 36 to 78 or the complementary sequencethereof, and which is capable of detecting the heat-resistant fungus;more preferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 1 to 23 and 36 to 78; or anoligonucleotide including the nucleotide sequence which has 70% or morehomology to the nucleotide sequence and which has a function as anoligonucleotide for detection; and still more preferably anoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 1 to 23 and 36 to 78.

Specifically, when identifying/detecting the fungi belonging to thegenus Byssochlamys of the heat-resistant fungi, it is preferable toperform gene amplification of a nucleic acid consisting of the whole orpart of the region of the above nucleic acid (A-I) or (A-II), and thento confirm whether the amplification product is present or not. In thiscase, as the nucleic acid primers, it is preferable to use anoligonucleotide which is capable of hybridizing with a region in thenucleic acid (A-I) or (A-II) and satisfies the following fourconditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Byssochlamys inthe nucleic acid (A-I) or (A-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value of about 55° C. to 65° C.

Further, it is preferable to use an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 1, 2 and 36 to39, or the complementary sequence thereof; or an oligonucleotideincluding the nucleotide sequence which has 70% or more homology to thenucleotide sequence set forth in any one of SEQ ID NOS: 1, 2 and 36 to39 or the complementary sequence thereof, and which has a function as anoligonucleotide for detection; more preferably an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 1,2 and 36 to 39; or an oligonucleotide including the nucleotide sequencewhich has 70% or more homology to the nucleotide sequence and which hasa function as an oligonucleotide for detection; and still morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 1, 2 and 36 to 39.

When identifying/detecting the fungi belonging to the genus Talaromycesof the heat-resistant fungi, it is preferable to perform geneamplification of a nucleic acid consisting of the whole or part of theregion of the above nucleic acid (B-I) or (B-II), and then to confirmwhether the amplification product is present or not. In this case, asthe nucleic acid primers, it is preferable to use an oligonucleotidewhich is capable of hybridizing with a region in the nucleic acid (B-I)or (B-II) and satisfies the following four conditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Talaromyces inthe nucleic acid (B-I) or (B-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value of about 55° C. to 65° C.

Further, it is preferable to use an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 3 to 11 and 57to 78, or the complementary sequence thereof; or an oligonucleotideincluding the nucleotide sequence which has 70% or more homology to thenucleotide sequence set forth in any one of SEQ ID NOS: 3 to 11 and 57to 78 or the complementary sequence thereof, and which has a function asan oligonucleotide for detection; more preferably an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 3to 11 and 57 to 78; or an oligonucleotide including the nucleotidesequence which has 70% or more homology to the nucleotide sequence andwhich has a function as an oligonucleotide for detection; and still morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 3 to 11 and 57 to 78.

When identifying/detecting the fungi belonging to the genus Neosartoryaand/or Aspergillus fumigatus of the heat-resistant fungi, it ispreferable to perform gene amplification of a nucleic acid consisting ofthe whole or part of the region of the above nucleic acid (C-I) or(C-II), and then to confirm whether the amplification product is presentor not. In this case, as the nucleic acid primers, it is preferable touse an oligonucleotide which is capable of hybridizing with a region inthe nucleic acid (C-I) or (C-II) and satisfies the following fourconditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Neosartoryaand/or Aspergillus fumigatus in the nucleic acid (C-I) or (C-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value of about 55° C. to 65° C.

Further, it is preferable to use an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 12 to 15, 22, 23and 40 to 50, or the complementary sequence thereof; or anoligonucleotide including the nucleotide sequence which has 70% or morehomology to the nucleotide sequence set forth in any one of SEQ ID NOS:12 to 15, 22, 23 and 40 to 50 or the complementary sequence thereof, andwhich has a function as an oligonucleotide for detection; morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 12 to 15, 22, 23 and 40 to 50, or anoligonucleotide including the nucleotide sequence which has 70% or morehomology to the nucleotide sequence and which has a function as anoligonucleotide for detection; and still more preferably anoligonucleotide including the nucleotide sequence set forth in any oneof SEQ ID NOS: 12 to 15, 22, 23 and 40 to 50.

When identifying/detecting the fungi belonging to the genus Hamigera ofthe heat-resistant fungi, it is preferable to perform gene amplificationof a nucleic acid consisting of the whole or part of the region of theabove nucleic acid (D-I) or (D-II), and then to confirm whether theamplification product is present or not. In this case, as the nucleicacid primers, it is preferable to use an oligonucleotide which iscapable of hybridizing with a region in the nucleic acid (D-I) or (D-II)and satisfies the following four conditions:

(1) the oligonucleotide includes a region containing about 10 continuousnucleotides specific to a fungus belonging to the genus Hamigera in thenucleic acid (D-I) or (D-II);

(2) the oligonucleotide has a GC content of about 30% to 80%;

(3) the oligonucleotide has low possibility to cause self-annealing; and

(4) the oligonucleotide has a Tm value of about 55° C. to 65° C.

Further, it is preferable to use an oligonucleotide including thenucleotide sequence set forth in any one of SEQ ID NOS: 16 to 21 and 51to 56, or the complementary sequence thereof; or an oligonucleotideincluding the nucleotide sequence which has 70% or more homology to thenucleotide sequence set forth in any one of SEQ ID NOS: 16 to 21 and 51to 56 or the complementary sequence thereof, and which has a function asan oligonucleotide for detection; more preferably an oligonucleotideincluding the nucleotide sequence set forth in any one of SEQ ID NOS: 16to 21 and 51 to 56; or an oligonucleotide including the nucleotidesequence which has 70% or more homology to the nucleotide sequence andwhich has a function as an oligonucleotide for detection; and still morepreferably an oligonucleotide including the nucleotide sequence setforth in any one of SEQ ID NOS: 16 to 21 and 51 to 56.

The method of amplifying the nucleic acid including the nucleotidesequence of the variable region is not particularly limited, and a usualmethod such as PCR (polymerase chain reaction) method, LCR (ligase chainreaction) method, SDA (strand displacement amplification) method, NASBA(nucleic acid sequence-based amplification) method, RCA (rolling-circleamplification) method, or LAMP (loop mediated isothermal amplification)method may be used. In the present invention, the PCR method or the LAMPmethod is preferably used in view of rapidness and ease as mentionedbelow.

As the nucleic acid primers for use in the present invention, theabove-mentioned oligonucleotides for detection of the present inventionmay be used without further treatment, or the oligonucleotides may belabeled with a labeling substance and used as nucleic acid primers.Examples of the labeling substance and labeling method include those inthe above case of the nucleic acid probes.

In the detection method of the present invention, amplificationreactions of any one of the nucleic acids (A-I) to (D-II) are preferablyperformed by a polymerase chain reaction (PCR) method.

Hereinafter, a detection method by the PCR method according the presentinvention is described in detail.

In the case where the fungi belonging to the genus Byssochlamys areidentified/detected by the PCR method, the following oligonucleotides(a) to (b) are preferably used as a nucleic acid primer, the followingoligonucleotides (a1) to (b1) are more preferably used as a nucleic acidprimer, and the oligonucleotides including the nucleotide sequence setforth in SEQ ID NO: 1 or 2 are still more preferably used as a nucleicacid primer.

Moreover, the following oligonucleotides (a) and (b) are preferably usedas a nucleic acid primer pair, the following oligonucleotides (a1) and(b1) are more preferably used as a nucleic acid primer pair, and theoligonucleotides including the nucleotide sequences set forth in SEQ IDNOS: 1 and 2 are still more preferably used as a nucleic acid primerpair.

(a) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 1 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(b) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 2 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(a1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 1, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(b1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 2, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

The oligonucleotides (a) and (b) can hybridize specifically with thevariable region of the β-tubulin gene of the fungi belonging to thegenus Byssochlamys. Therefore, it is possible to specifically, rapidly,and easily detect the fungi belonging to the genus Byssochlamys by usingthe oligonucleotides.

The oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 1 or 2 is an oligonucleotide complementary to a nucleotidesequence which is in the β-tubulin gene region and is specific to thefungi belonging to the genus Byssochlamys (i.e., oligonucleotidescomplementary to a part of the variable region). The oligonucleotidesincluding the nucleotide sequence set forth in SEQ ID NO: 1 or 2 canhybridize specifically with a part of DNA or RNA of the fungi belongingto the genus Byssochlamys.

The variable region in the β-tubulin gene of the fungus belonging to thegenus Byssochlamys is described in detail based on the variable regionof Byssochlamys nivea as an example. As mentioned above, the partialnucleotide sequence of the β-tubulin gene of Byssochlamys nivea isrepresented by SEQ ID NO: 24. The inventors of the present inventionhave found out that a nucleotide sequence of the region of position 20to 175 in the partial nucleotide sequence of the β-tubulin gene of thefungi belonging to the genus Byssochlamys is particularly poorlyconserved among fungi genera, and each of the genera has a specificnucleotide sequence in this region.

The oligonucleotides (a) and (b) correspond to the region of position 33to 52 and the region of position 159 to 178 in the nucleotide sequenceset forth in SEQ ID NO: 24, respectively. Therefore, it is possible tospecifically detect the fungi belonging to the genus Byssochlamys byhybridizing the oligonucleotides with the β-tubulin gene of the fungibelonging to the genus Byssochlamys.

In the case where the fungi belonging to the genus Talaromyces areidentified/detected by the PCR method, the following oligonucleotides(c) to (k) are preferably used as a nucleic acid primer, the followingoligonucleotides (c1) to (k1) are more preferably used as a nucleic acidprimer, and the oligonucleotides including the nucleotide sequence setforth in any one of SEQ ID NOS: 3 to 11 are still more preferably usedas a nucleic acid primer.

Moreover, the oligonucleotide pair (c) and (d), the oligonucleotide pair(e) and (f), the oligonucleotide pair (g) and (h), the oligonucleotidepair (i) and (h), and the oligonucleotide pair (j) and (k) arepreferably used as a nucleic acid primer pair; the oligonucleotide pair(c1) and (d1), the oligonucleotide pair (e1) and (f1), theoligonucleotide pair (g1) and (h1), the oligonucleotide pair (i1) and(h1), and the oligonucleotide pair (j1) and (k1) are more preferablyused as a nucleic acid primer pair; and the oligonucleotide pairincluding the nucleotide sequences set forth in SEQ ID NOS: 3 and 4, theoligonucleotide pair including the nucleotide sequences set forth in SEQID NOS: 5 and 6, the oligonucleotide pair including the nucleotidesequences set forth in SEQ ID NOS: 7 and 8, the oligonucleotide pairincluding the nucleotide sequences set forth in SEQ ID NOS: 9 and 8, andthe oligonucleotide pair including the nucleotide sequences set forth inSEQ ID NOS: 10 and 11 are still more preferably used as a nucleic acidprimer pair.

In view of detection specificity and detection sensitivity, theoligonucleotide pair (i) and (h), and the oligonucleotide pair (j) and(k) are preferably used as a nucleic acid primer pair; theoligonucleotide pair (i1) and (h1), and the oligonucleotide pair (j1)and (k1) are more preferably used as a nucleic acid primer pair; and theoligonucleotide pair including the nucleotide sequences set forth in SEQID NOS: 9 and 8, and the oligonucleotide pair including the nucleotidesequences set forth in SEQ ID NOS: 10 and 11 are still more preferablyused as a nucleic acid primer pair.

(c) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 3 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(d) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 4 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(e) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 5 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(f) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 6 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(g) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 7 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(h) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 8 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(i) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 9 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(j) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 10 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(k) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 11 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(c1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 3, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(d1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 4, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(e1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 5, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(f1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 6, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(g1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 7, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(h1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 8, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(i1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 9, or a nucleotide sequence which has 70% or more homology to thenucleotide sequence and which has a function as an oligonucleotide fordetection.

(j1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 10, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(k1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 11, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

When the pair of the oligonucleotides (c) and (d) is used, Talaromycesflavus and Talaromyces trachyspermus can be specifically detected. Whenthe pair of the oligonucleotides (e) and (f) or the pair of theoligonucleotides (j) and (k) is used, Talaromyces luteus, Talaromyceswortmannii and Talaromyces bacillisporus can be specifically detected.When the pair of the oligonucleotides (g) and (h) is used, Talaromycesflavus, Talaromyces wortmannii, Talaromyces trachyspermus andTalaromyces macrosporus can be specifically detected. When the pair ofthe oligonucleotides (i) and (h) is used, Talaromyces flavus,Talaromyces trachyspermus, and Talaromyces macrosporus can bespecifically detected.

The oligonucleotides (c) to (k) can hybridize specifically with thevariable region in the β-tubulin gene or the ITS region and D1/D2 regionof 28S rDNA of the fungi belonging to the genus Talaromyces. Therefore,it is possible to specifically, rapidly, and easily detect the fungibelonging to the genus Talaromyces by using the oligonucleotides.

The oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 3 or 4 is an oligonucleotide complementary to a nucleotidesequence which is in the β-tubulin gene region and is specific toTalaromyces flavus and Talaromyces trachyspermus belonging to the genusTalaromyces (i.e., oligonucleotides complementary to a part of thevariable region). The oligonucleotide represented by the nucleotidesequence set forth in any one of SEQ ID NOS: 5 to 6 and 10 to 11 is anoligonucleotide complementary to a nucleotide sequence which is in theβ-tubulin gene region and is specific to Talaromyces luteus, Talaromyceswortmannii and Talaromyces bacillisporus belonging to the genusTalaromyces (i.e., oligonucleotides complementary to a part of thevariable region). The oligonucleotide represented by the nucleotidesequence set forth in any one of SEQ ID NOS: 7 to 8 and 9 is anoligonucleotide complementary to a nucleotide sequence which is in theITS region and D1/D2 region of 28S rDNA and is specific to Talaromycesflavus, Talaromyces wortmannii, Talaromyces trachyspermus andTalaromyces macrosporus belonging to the genus Talaromyces (i.e.,oligonucleotides complementary to a part of the variable region). Thatis, the oligonucleotides including the nucleotide sequence set forth inany one of SEQ ID NOS: 3 to 11 can hybridize specifically with a part ofDNA or RNA of the fungi belonging to the genus Talaromyces.

The variable region in the β-tubulin gene of the fungus belonging to thegenus Talaromyces is described in detail based on the variable region ofTalaromyces flavus and Talaromyces luteus as examples. As mentionedabove, the partial nucleotide sequence of the β-tubulin gene ofTalaromyces flavus is represented by SEQ ID NO: 26. The partialnucleotide sequence of the β-tubulin gene of Talaromyces luteus isrepresented by SEQ ID NO: 27. The inventors of the present inventionhave found out that nucleotide sequences of the region of position 10 to40 and the region of position 70 to 100 in the partial nucleotidesequence of the β-tubulin gene (the variable region) of Talaromycesflavus are particularly highly conserved in Talaromyces flavus andTalaromyces trachyspermus, and are not similar to any sequences of otherfungi. The inventors of the present invention also have found out thatthe region of position 120 to 160 and the region of position 295 to 325in the partial nucleotide sequence of the β-tubulin gene (the variableregion) of Talaromyces luteus are regions having sequences highlyconserved in genetically related Talaromyces luteus, Talaromyceswortmannii, and Talaromyces bacillisporus, and are not similar to anysequences of other fungi.

The oligonucleotides (c) and (d) correspond to the region of position 15to 34 and the region of position 76 to 98 in the nucleotide sequence setforth in SEQ ID NO: 26, respectively. The oligonucleotides (e) and (f),and (j) and (k) correspond to the region of position 133 to 153 and theregion of position 304 to 325 in the nucleotide sequence set forth inSEQ ID NO: 27, respectively. Therefore, it is possible to specificallydetect the fungi belonging to the genus Talaromyces by hybridizing theoligonucleotides with the β-tubulin gene of the fungi belonging to thegenus Talaromyces.

The variable region in the ITS region and D1/D2 region of 28S rDNA ofthe fungus belonging to the genus Talaromyces is described in detailbased on the variable region of Talaromyces wortmannii as an example. Asmentioned above, the partial nucleotide sequence of the ITS region andD1/D2 region of 28S rDNA of Talaromyces wortmannii is represented by SEQID NO: 28. The inventors of the present invention have found out thatnucleotide sequences of the region of position 300 to 350 and the regionof position 450 to 510 in the partial nucleotide sequence of the ITSregion and D1/D2 region of 28S rDNA of the fungi belonging to the genusTalaromyces are particularly highly conserved in Talaromyces wortmannii,Talaromyces trachyspermus, Talaromyces flavus and Talaromycesmacrosporus, and are not similar to any sequences of other fungi.

The oligonucleotides (g), (h) and (i) correspond to the region ofposition 326 to 345 and the region of position 460 to 478 in thenucleotide sequence set forth in SEQ ID NO: 28, respectively. Therefore,it is possible to specifically detect the fungi belonging to the genusTalaromyces by hybridizing the oligonucleotides with the ITS region andD1/D2 region of 28S rDNA of the fungi belonging to the genusTalaromyces.

In the case where the fungi belonging to the genus Neosartorya and/orAspergillus fumigatus are identified/detected by the PCR method, thefollowing oligonucleotides (l) to (o) and (v) to (w) are preferably usedas a nucleic acid primer, the following oligonucleotides (l1) to (o1)and (v1) to (w1) are more preferably used as a nucleic acid primer, andthe oligonucleotides including the nucleotide sequence set forth in anyone of SEQ ID NOS: 12 to 15 and 22 to 23 are still more preferably usedas a nucleic acid primer.

Moreover, the oligonucleotide pair (l) and (m), the oligonucleotide pair(n) and (o), and the oligonucleotide pair (v) and (w) are preferablyused as a nucleic acid primer pair; the oligonucleotide pair (l1) and(m1), the oligonucleotide pair (n1) and (o1), and the oligonucleotidepair (v1) and (w1) are more preferably used as a nucleic acid primerpair; and the oligonucleotide pair including the nucleotide sequencesset forth in SEQ ID NOS: 12 and 13, the oligonucleotide pair includingthe nucleotide sequences set forth in SEQ ID NOS: 14 and 15, and theoligonucleotide pair including the nucleotide sequences set forth in SEQID NOS: 22 and 23 are still more preferably used as a nucleic acidprimer pair.

For detecting the fungi belonging to the genus Neosartorya andAspergillus fumigatus, the oligonucleotide pair (l) and (m), and theoligonucleotide pair (n) and (o) are preferably used as a nucleic acidprimer pair; the oligonucleotide pair (l1) and (m1), and theoligonucleotide pair (n1) and (o1) are more preferably used as a nucleicacid primer pair; and the oligonucleotide pair including the nucleotidesequences set forth in SEQ ID NOS: 12 and 13, and the oligonucleotidepair including the nucleotide sequences set forth in SEQ ID NOS: 14 and15 are still more preferably used as a nucleic acid primer pair.Further, in view of detection specificity and detection sensitivity, theoligonucleotide pair (n) and (o) are preferably used as a nucleic acidprimer pair; the oligonucleotide pair (n1) and (o1) are more preferablyused as a nucleic acid primer pair; and the oligonucleotide pairincluding the nucleotide sequences set forth in SEQ ID NOS: 14 and 15are still more preferably used as a nucleic acid primer pair.

For detecting Aspergillus fumigatus, the oligonucleotide pair (v) and(w) are preferably used as a nucleic acid primer pair; theoligonucleotide pair (v1) and (w1) are more preferably used as a nucleicacid primer pair; and the oligonucleotide pair including the nucleotidesequences set forth in SEQ ID NOS: 22 and 23 are still more preferablyused as a nucleic acid primer pair.

(l) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 12 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(m) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 13 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(n) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 14 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(o) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 15 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(v) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 22 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(w) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 23 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(l1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 12, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(m1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 13, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(n1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 14, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(o1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 15, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(v1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 22, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(w1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 23, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

When the pair of the oligonucleotides (l) and (m) and the pair of theoligonucleotides (n) and (o) are used, the fungus belonging to the genusNeosartorya such as Neosartorya fischeri, Neosartorya spinosa,Neosartorya glabra, Neosartorya hiratsukae, Neosartorya paulistensis,Neosartorya pseudo fischeri, and Aspergillus fumigatus can bespecifically detected. When the pair of the oligonucleotides (v) and (w)is used, Aspergillus fumigatus can be specifically detected.

The oligonucleotides (l) to (o) can hybridize specifically with thevariable region in the β-tubulin gene of the fungi belonging to thegenus Neosartorya and Aspergillus fumigatus. Therefore, it is possibleto specifically, rapidly, and easily detect the fungi belonging to thegenus Neosartorya and Aspergillus fumigatus by using theoligonucleotides (l) to (o).

The oligonucleotide represented by the nucleotide sequence set forth inany one of SEQ ID NOS: 12 to 15 is an oligonucleotide complementary to anucleotide sequence which is in the β-tubulin gene region and isspecific to Neosartorya fischeri, Neosartorya spinosa, Neosartoryaglabra, Neosartorya hiratsukae, Neosartorya paulistensis, Neosartoryapseudofischeri and the like which belong to the genus Neosartorya, andAspergillus fumigatus (i.e., oligonucleotides complementary to a part ofthe variable region). The oligonucleotides can hybridize specificallywith a part of DNA or RNA of the fungi belonging to the genusNeosartorya and Aspergillus fumigatus.

The variable region in the β-tubulin gene of the fungus belonging to thegenus Neosartorya is described in detail based on the variable region ofNeosartorya glabra as an example. As mentioned above, the partialnucleotide sequence of the β-tubulin gene of Neosartorya glabra isrepresented by SEQ ID NO: 32. The inventors of the present inventionhave found out that nucleotide sequences of the region of position 1 to110, the region of position 140 to 210 and the region of position 350 to380 in the partial nucleotide sequence of the β-tubulin gene of thefungi belonging to the genus Neosartorya are particularly poorlyconserved among fungi, and each of fungi genera has a specificnucleotide sequence in this region. The partial nucleotide sequence ofthe β-tubulin gene of Aspergillus fumigatus is represented by SEQ ID NO:33. The inventors of the present invention have found out thatnucleotide sequences of the region of position 1 to 110, the region ofposition 140 to 210 and the region of position 350 to 380 in the partialnucleotide sequence of the β-tubulin gene of Aspergillus fumigatus areparticularly poorly conserved among fungi, and each of fungi species hasa specific nucleotide sequence in this region.

The oligonucleotides (l) and (m) correspond to the region of position 84to 103 and the region of position 169 to 188 in the nucleotide sequenceset forth in SEQ ID NO: 32, and the region of position 83 to 102 and theregion of position 166 to 186 in the nucleotide sequence set forth inSEQ ID NO: 33, respectively. The oligonucleotides (n) and (o) correspondto the region of position 144 to 163 and the region of position 358 to377 in the nucleotide sequence set forth in SEQ ID NO: 32, and theregion of position 141 to 160 and the region of position 356 to 376 inthe nucleotide sequence set forth in SEQ ID NO: 33, respectively.Therefore, it is possible to specifically detect the fungi belonging tothe genus Neosartorya and Aspergillus fumigatus by hybridizing theoligonucleotides with the β-tubulin gene of the fungi belonging to thegenus Neosartorya and/or Aspergillus fumigatus.

The oligonucleotides (v) and (w) can hybridize specifically with thevariable region in the β-tubulin gene of Aspergillus fumigatus.

The oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 22 or 23 is an oligonucleotide complementary to a nucleotidesequence which is in the β-tubulin gene region and is specific toAspergillus fumigatus (i.e., oligonucleotides complementary to a part ofthe variable region). Therefore, the oligonucleotides including thenucleotide sequence set forth in SEQ ID NO: 22 or 23 can hybridizespecifically with a part of DNA or RNA of Aspergillus fumigatus, butcannot hybridize with DNA and RNA of the fungi belonging to the genusNeosartorya.

Other variable region in the β-tubulin gene of Aspergillus fumigatus isdescribed. The partial nucleotide sequence of the β-tubulin gene ofAspergillus fumigatus is represented by SEQ ID NO: 33. The inventors ofthe present invention have found out that the region of position 20 to50 and the region of position 200 to 230 in the partial nucleotidesequence of the β-tubulin gene of Aspergillus fumigatus are poorlyconserved among fungi, in particular, the fungi belonging to the genusNeosartorya.

The oligonucleotides (v) and (w) correspond to the region of position 23to 44 and the region of position 200 to 222 in the nucleotide sequenceset forth in SEQ ID NO: 33, respectively. The oligonucleotides canhybridize with the β-tubulin gene of Aspergillus fumigatus but cannothybridize with DNA and RNA of the fungi belonging to the genusNeosartorya. The fact is described in detail based on FIG. 1. FIG. 1 isa diagram for comparing partial nucleotide sequences of the β-tubulingenes of Aspergillus fumigatus, Neosartorya fischeri, and Neosartoryaspinosa set forth in SEQ ID NOS: 34 and 83 to 86. As shown in FIG. 1, acomparison between the region which is in the β-tubulin gene ofAspergillus fumigatus and is recognized by the oligonucleotides of SEQID NOS: 22 and 23 and the region which corresponds to theabove-mentioned region and is in the β-tubulin gene of the fungibelonging to the genus Neosartorya reveals that the homology of thenucleotide sequences are very low compared with other regions.Therefore, when the oligonucleotides (v) and (w) are used, it ispossible to discriminate Aspergillus fumigatus from the fungus belongingto the genus Neosartorya in a sample.

An example of detecting the fungi belonging to the genus Neosartoryawhich particularly cause problems in food accidents is described below.The fungus belonging to the genus Neosartorya and Aspergillus fumigatusin samples can be detected by performing a nucleic acid amplificationtreatment using the pair of the oligonucleotides (l) and (m) or the pairof the oligonucleotides (n) and (o) as nucleic acid primers, and thenconfirming gene amplification. The samples from which the fungibelonging to the genus Neosartorya and Aspergillus fumigatus have beendetected by the above process are further subjected to a nucleic acidamplification treatment using the pair of the oligonucleotides (v) and(w) as nucleic acid primers, followed by confirmation of geneamplification. As a result, it is possible to discriminate Aspergillusfumigatus from the fungus belonging to the genus Neosartorya in thesample.

Further, it is also possible to discriminate/detect the species of fungiin samples which show positive results by the above detection methodusing the oligonucleotides (l) to (o), based on a difference in growthtemperature zones of the fungi belonging to the genus Neosartorya andAspergillus fumigatus or by using the oligonucleotides (v) and/or (w).

The upper limit of the growth temperature of the fungi belonging to thegenus Neosartorya is about 45° C. On the other hand, Aspergillusfumigatus can grow at 50° C. or higher. Based on the difference in thegrowth temperature zones, for example, the following procedure may beperformed to discriminate between the species of fungi. However, thepresent invention is not limited thereto.

For samples which show positive results by the detection method usingthe oligonucleotides (l) to (o), hyphae from single colonies areinoculated into a PDA medium, PDB medium (potato dextrose liquid medium)or the like. Culture is performed at 48 to 52° C. for one or two days,and elongation of the hyphae is confirmed. Then, based on the differencein the growth temperature zones, the fungus can be discriminated asAspergillus fumigatus in the case where proliferation is confirmed, orthe fungus can be discriminated as a fungus belonging to the genusNeosartorya in the case where proliferation is not confirmed. It shouldbe noted that examples of a method of confirming proliferation include,but not limited to, by confirming elongation of hyphae by astereomicroscope, elongation of hyphae in a liquid medium, formation offungal granules, and formation of conidia.

In the case where the fungi belonging to the genus Hamigera areidentified/detected by the PCR method, the following oligonucleotides(p) to (u) are preferably used as a nucleic acid primer, the followingoligonucleotides (p1) to (u1) are more preferably used as a nucleic acidprimer, and the oligonucleotides including the nucleotide sequence setforth in any one of SEQ ID NOS: 16 to 21 are still more preferably usedas a nucleic acid primer.

Moreover, the oligonucleotide pair (p) and (q), the oligonucleotide pair(r) and (s), and the oligonucleotide pair (t) and (u) are preferablyused as a nucleic acid primer pair; the oligonucleotide pair (p1) and(q1), the oligonucleotide pair (r1) and (s1), and the oligonucleotidepair (t1) and (u1) are more preferably used as a nucleic acid primerpair; and the oligonucleotide pair including the nucleotide sequencesset forth in SEQ ID NOS: 16 and 17, the oligonucleotide pair includingthe nucleotide sequences set forth in SEQ ID NOS: 18 and 19, and theoligonucleotide pair including the nucleotide sequences set forth in SEQID NOS: 20 and 21 are still more preferably used as a nucleic acidprimer pair.

In view of detection specificity, the oligonucleotide pair (r) and (s),and the oligonucleotide pair (t) and (u) are preferably used as anucleic acid primer pair; the oligonucleotide pair (r1) and (s1), andthe oligonucleotide pair (t1) and (u1) are more preferably used as anucleic acid primer pair; and the oligonucleotide pair including thenucleotide sequences set forth in SEQ ID NOS: 18 and 19, and theoligonucleotide pair including the nucleotide sequences set forth in SEQID NOS: 20 and 21 are still more preferably used as a nucleic acidprimer pair. In view of detection sensitivity, the oligonucleotide pair(t) and (u) are preferably used as a nucleic acid primer pair; theoligonucleotide pair (t1) and (u1) are more preferably used as a nucleicacid primer pair; and the oligonucleotide pair including the nucleotidesequences set forth in SEQ ID NOS: 20 and 21 are still more preferablyused as a nucleic acid primer pair.

(p) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 16 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(q) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 17 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(r) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 18 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(s) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 19 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(t) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 20 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(u) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 21 or a complementary sequence thereof; or a nucleotide sequencewhich has 70% or more homology to the nucleotide sequence or thecomplementary sequence thereof, and which has a function as anoligonucleotide for detection.

(p1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 16, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(q1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 17, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(r1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 18, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(s1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 19, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(t1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 20, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

(u1) An oligonucleotide including a nucleotide sequence set forth in SEQID NO: 21, or a nucleotide sequence which has 70% or more homology tothe nucleotide sequence and which has a function as an oligonucleotidefor detection.

When the pair of the oligonucleotides (p) and (q), the pair of theoligonucleotides (r) and (s), and the pair of the oligonucleotides (t)and (u) are used, Hamigera avellanea and Hamigera striata can bespecifically detected.

The oligonucleotides (p) to (u) can hybridize specifically with thevariable region in the β-tubulin gene of the fungi belonging to thegenus Hamigera. Therefore, it is possible to specifically, rapidly, andeasily detect the fungi belonging to the genus Hamigera by using theoligonucleotides.

The oligonucleotide represented by the nucleotide sequence set forth inany one of SEQ ID NOS: 16 to 21 is an oligonucleotide complementary to anucleotide sequence which is in the β-tubulin gene region and isspecific to the fungi belonging to the genus Hamigera (i.e.,oligonucleotides complementary to a part of the variable region). Theoligonucleotides including the nucleotide sequence set forth in any oneof SEQ ID NOS: 16 to 21 can hybridize specifically with a part of DNA orRNA of the fungi belonging to the genus Hamigera.

The variable region in the β-tubulin gene of the fungus belonging to thegenus Hamigera is described in detail based on the variable region ofHamigera avellanea as an example. As mentioned above, the partialnucleotide sequence of the β-tubulin gene of Hamigera avellanea isrepresented by SEQ ID NO: 35. The inventors of the present inventionhave found out that nucleotide sequences of the region of position 350to 480, the region of position 1 to 25, and the region of position 180to 2080 in the partial nucleotide sequence of the β-tubulin gene of thefungi belonging to the genus Hamigera are particularly poorly conservedamong fungi genera, and each of the genera has a specific nucleotidesequence in this region.

The oligonucleotides (p) and (q) correspond to the region of position358 to 377 and the region of position 440 to 459 in the nucleotidesequence set forth in SEQ ID NO: 35, respectively. The oligonucleotides(r) and (s) correspond to the region of position 2 to 22 and the regionof position 181 to 200 in the nucleotide sequence set forth in SEQ IDNO: 35, respectively. The oligonucleotides (t) and (u) correspond to theregion of position 172 to 191 and the region of position 397 to 416 inthe nucleotide sequence set forth in SEQ ID NO: 35, respectively.Therefore, it is possible to specifically detect the fungi belonging tothe genus Hamigera by hybridizing the oligonucleotides with theβ-tubulin gene of the fungi belonging to the genus Hamigera.

It should be noted that, as shown in FIG. 2, regions having highhomology to the regions of position 358 to 377 and position 440 to 459of the β-tubulin gene of Hamigera avellanea are present in the β-tubulingene of a fungus belonging to the genus Cladosporium such asCladosporium cladosporoides. However, a region having high homology tothe region of position 181 to 200 of the β-tubulin gene of Hamigeraavellanea is not present in the β-tubulin gene of the fungus belongingto the genus Cladosporium. Therefore, when the above-mentionedoligonucleotides (p) to (u) are used in combination, it is possible todetect fungi belonging to the genus Hamigera and fungi belonging to thegenus Cladosporium.

That is, the oligonucleotides (s), (t), and (u) can hybridize with thevariable region in the β-tubulin gene of the fungi belonging to thegenus Hamigera but cannot hybridize with the variable region in theβ-tubulin gene of the fungi belonging to the genus Cladosporium. Forexample, for samples which show positive results by the detection methodusing the oligonucleotides (p) and (q), the species of the fungi in thesamples can be discriminated as the genus Hamigera or the genusCladosporium by using the oligonucleotides (r) and (s) and/or theoligonucleotides (t) and (u).

The “fungi belonging to the genus Cladosporium” are filamentousdeuteromycetes and do not form highly heat-resistant ascospores. Inaddition, the fungi are widely distributed in places to live or foodfactories and synthesize melanin pigment, and are fungi causing blackstains. Examples of the fungi belonging to the genus Cladosporiuminclude Cladosporium cladosporoides and Cladosporium sphaerospermum.

Conditions of the PCR reaction are not particularly limited as long as aDNA fragment of interest can be amplified to a detectable degree. Apreferred example of the conditions of PCR reaction is as follows. Whendetecting the fungus belonging to the genus Byssochlamys, a cycleincluding: a thermal denaturation reaction for denaturation ofdouble-stranded DNA into single strands at 95 to 98° C. for 10 to 60seconds; an annealing reaction for hybridization of a primer pair withthe single-stranded DNA at about 59 to 61° C. for about 60 seconds; andan elongation reaction for a reaction of a DNA polymerase at about 72°C. for about 60 seconds; is repeated about 30 to 35 times. Whendetecting the fungus belonging to the genus Talaromyces, a cycleincluding: a thermal denaturation reaction for denaturation ofdouble-stranded DNA into single strands at 95 to 97° C. for 10 to 60seconds; an annealing reaction for hybridization of a primer pair withthe single-stranded DNA at about 55 to 61° C. for about 60 seconds; andan elongation reaction for a reaction of a DNA polymerase at about 72°C. for about 60 seconds; is repeated about 30 to 35 times. In the casewhere the oligonucleotides (l) and (m), or (n) and (o) are used todetect the fungus belonging to the genus Neosartorya and Aspergillusfumigatus, a cycle including: a thermal denaturation reaction fordenaturation of double-stranded DNA into single strands at 95 to 98° C.for 10 to 60 seconds; an annealing reaction for hybridization of aprimer pair with the single-stranded DNA at about 59 to 61° C. for about60 seconds; and an elongation reaction for a reaction of a DNApolymerase at about 72° C. for about 60 seconds; is repeated about 30 to35 times. In the case where the oligonucleotides (v) and (w) are used todetect whether a fungus is the fungus belonging to the genus Neosartoryaor Aspergillus fumigatus, a cycle including: a thermal denaturationreaction for denaturation of double-stranded DNA into single strands at95 to 98° C. for 10 to 60 seconds; an annealing reaction forhybridization of a primer pair with the single-stranded DNA at about 59to 61° C. for about 60 seconds; and an elongation reaction for areaction of a DNA polymerase at about 72° C. for about 60 seconds; isrepeated about 30 to 35 times. In the case where the oligonucleotides(p) and (q) are used to detect the fungus belonging to the genusHamigera, a cycle including: a thermal denaturation reaction fordenaturation of double-stranded DNA into single strands at 95 to 98° C.for 10 to 60 seconds; an annealing reaction for hybridization of aprimer pair with the single-stranded DNA at about 59 to 63° C. for about60 seconds; and an elongation reaction for a reaction of a DNApolymerase at about 72° C. for about 60 seconds; is repeated about 30 to35 times. In the case where the oligonucleotides (r) and (s), or (t) and(u) are used to detect the fungus belonging to the genus Hamigera, acycle including: a thermal denaturation reaction for denaturation ofdouble-stranded DNA into single strands at 95 to 98° C. for 10 to 60seconds; an annealing reaction for hybridization of a primer pair withthe single-stranded DNA at about 59 to 63° C. for about 60 seconds; andan elongation reaction for a reaction of a DNA polymerase at about 72°C. for about 60 seconds; is repeated about 30 to 35 times.

In the present invention, amplification of gene fragments may beconfirmed by a usual method. Examples of the method include, but notlimited to, a method of integrating nucleotides labeled with aradioactive substance or the like into reaction products during anamplification reaction, a method including performing electrophoresisfor PCR reaction products and confirming the existence of a bandcorresponding to the size of the amplified gene, a method of determiningnucleotide sequences of PCR reaction products, and a method ofintegrating fluorescent substances into between the double strands ofamplified DNA. In the present invention, the method including performingelectrophoresis after a gene amplification treatment and confirming theexistence of a band corresponding to the size of the amplified gene ispreferred.

At position 33 to 178 in the nucleotide sequence set forth in SEQ ID NO:24, the number of nucleotides is 146. Therefore, in the case where asample contains the fungus belonging to the genus Byssochlamys, DNAfragments of about 150 bp which are specific to the fungi belonging tothe genus Byssochlamys can be detected by performing PCR reactions usingthe pair of the oligonucleotides (a) and (b) as a primer set andelectrophoresing the resultant PCR reaction products. By doing the aboveprocedure, the fungi belonging to the genus Byssochlamys can be detectedor discriminated.

At position 15 to 98 in the nucleotide sequence set forth in SEQ ID NO:26, the number of nucleotides is 83. At position 133 to 325 in thenucleotide sequence set forth in SEQ ID NO: 27, the number ofnucleotides is 192. Meanwhile, at position 326 to 478 in the nucleotidesequence set forth in SEQ ID NO: 28, the number of nucleotides is 152.Therefore, in the case where a sample contains Talaromyces flavus and/orTalaromyces trachyspermus, DNA fragments of about 80 bp which arespecific to the fungi can be detected by performing PCR reactions usingthe pair of the oligonucleotides (c) and (d) as a primer set andelectrophoresing the resultant PCR reaction products. In the case wherea sample contains Talaromyces luteus, Talaromyces wortmannii and/orTalaromyces bacillisporus, DNA fragments of about 200 bp which arespecific to the fungi can be detected by performing PCR reactions usingthe pair of the oligonucleotides (e) and (f) or the pair of theoligonucleotides (j) and (k) as a primer set and electrophoresing theresultant PCR reaction products. In the case where a sample containsTalaromyces macrosporus, Talaromyces wortmannii, Talaromyces flavusand/or Talaromyces trachyspermus, DNA fragments of about 150 bp whichare specific to the fungi can be detected by performing PCR reactionsusing the pair of the oligonucleotides (g) and (h) as a primer set andelectrophoresing the resultant PCR reaction products. In the case wherea sample contains Talaromyces macrosporus, Talaromyces flavus and/orTalaromyces trachyspermus, DNA fragments of about 150 bp which arespecific to the fungi can be detected by performing PCR reactions usingthe pair of the oligonucleotides (i) and (h) as a primer set andelectrophoresing the resultant PCR reaction products. By doing theabove-mentioned procedure, the fungi belonging to the genus Talaromycescan be detected or discriminated. It should be noted that, in the casewhere a sample contains Talaromyces wortmannii, two bands correspondingto about 200 bp and about 150 bp are confirmed by simultaneously usingthe pair of the oligonucleotides (e) and (f) or the oligonucleotides (j)and (k), and the pair of the oligonucleotides (g) and (h).

In the case where a sample contains the fungus belonging to the genusNeosartorya and/or Aspergillus fumigatus, DNA fragments of about 100 bpwhich are specific to the fungi can be detected by performing PCRreactions using the pair of the oligonucleotides (l) and (m) as a primerset and electrophoresing the resultant PCR reaction products. Also, DNAfragments of about 200 bp which are specific to the fungi can bedetected by performing PCR reactions using the pair of theoligonucleotides (n) and (o) as a primer set and electrophoresing theresultant PCR reaction products. By doing the above-mentioned procedure,the fungi belonging to the genus Neosartorya and Aspergillus fumigatuscan be detected or discriminated.

Further, in the case where a sample contains Aspergillus fumigatus, DNAfragments of about 200 bp which are specific to Aspergillus fumigatuscan be detected by performing PCR reactions using the pair of theoligonucleotides (v) and (w) as a primer set and electrophoresing theresultant PCR reaction products. On the other hand, in the case where asample contains the fungi belonging to the genus Neosartorya, even ifPCR reactions are performed using the pair of the oligonucleotides (v)and (w), amplification of DNA fragments cannot be observed. Therefore,when PCR reactions are performed using the pair of the oligonucleotides(v) and (w) as a primer set, only Aspergillus fumigatus can be detected.

In the case where the pair of the oligonucleotides (p) and (q) are usedto confirm gene amplification, the number of nucleotides at position 358to 459 in the nucleotide sequence set forth in SEQ ID NO: 35 is about100. Therefore, in the case where a sample contains the fungus belongingto the genus Hamigera, DNA fragments of about 100 bp which are specificto the fungi can be detected by performing PCR reactions using the pairof the oligonucleotides (p) and (q) as a primer set and electrophoresingthe resultant PCR reaction products.

The number of nucleotides at position 2 to 200 in the nucleotidesequence set forth in SEQ ID NO: 35 is about 200. In the case where thepair of the oligonucleotides (t) and (u) are used, the number ofnucleotides at position 172 to 416 in the nucleotide sequence set forthin SEQ ID NO: 35 is about 245. Therefore, in the case where a samplecontains the fungus belonging to the genus Hamigera, DNA fragments ofabout 200 bp or about 240 bp which are specific to the fungi can bedetected by performing PCR reactions using the pair of theoligonucleotides (r) and (s) or the pair of the oligonucleotides (t) and(u) as a primer set and electrophoresing the resultant PCR reactionproducts. By doing the above-mentioned procedure, the fungi belonging tothe genus Hamigera can be detected or discriminated.

In the detection method of the present invention it is preferable tosimultaneously use a detection method using the pair of theoligonucleotides (c) and (d), the pair of the oligonucleotides (g) and(h), or the pair of the oligonucleotides and (i) and (h) and a detectionmethod using the pair of the oligonucleotides (e) and (f) or the pair ofthe oligonucleotides (j) and (k), for detecting the fungi belonging tothe genus Talaromyces. When a plurality of pairs of the oligonucleotidesis used in combination, the fungi belonging to the genus Talaromyces canbe exhaustively detected. In particular, it is more preferable to use adetection method using the pair of the oligonucleotides (c1) and (d1),the pair of the oligonucleotides (g1) and (h1), or the pair of theoligonucleotides (i1) and (h1) and a detection method using the pair ofthe oligonucleotides (e1) and (f1) or the pair of the oligonucleotides(j1) and (k1) in combination, and it is still more preferable to use adetection method using the pair of the oligonucleotides represented bythe nucleotide sequences set forth in SEQ ID NOS: 3 and 4, the pair ofthe oligonucleotides represented by the nucleotide sequences set forthin SEQ ID NOS: 7 and 8, or the pair of the oligonucleotides representedby the nucleotide sequences set forth in SEQ ID NOS: 9 and 8 and adetection method using the pair of the oligonucleotides represented bythe nucleotide sequences set forth in SEQ ID NOS: 5 and 6 or the pair ofthe oligonucleotides represented by the nucleotide sequences set forthin SEQ ID NOS: 10 and 11 in combination.

When the pair of the oligonucleotides (c) and (d) is used, Talaromycesflavus and Talaromyces trachyspermus can be specifically detected. Whenthe pair of the oligonucleotides (e) and (f) or the pair of theoligonucleotides (j) and (k) is used, Talaromyces luteus, Talaromyceswortmannii and Talaromyces bacillisporus can be specifically detected.When the pair of the oligonucleotides (g) and (h) is used, Talaromycesflavus, Talaromyces wortmannii, Talaromyces trachyspermus andTalaromyces macrosporus can be specifically detected. When the pair ofthe oligonucleotides (i) and (h) is used, Talaromyces flavus,Talaromyces trachyspermus, and Talaromyces macrosporus can bespecifically detected. Therefore, when the pair of the oligonucleotides(c) and (d), the pair of the oligonucleotides (g) and (h), or the pairof the oligonucleotides (i) and (h) is used in combination with the pairof the oligonucleotides (e) and (f) or the pair of the oligonucleotides(j) and (k), the fungi belonging to the genus Talaromyces whichparticularly cause problems in food accidents can be exhaustivelydetected.

In particular, in view of detection sensitivity, it is preferred to usea detection method using the pair of oligonucleotides (i) and (h) and adetection method using the pair of oligonucleotides (j) and (k) incombination, it is more preferred to use a detection method using thepair of oligonucleotides (i1) and (h1) and a detection method using thepair of oligonucleotides (j1) and (k1) in combination, and it is stillmore preferred to use a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 9 and 8 and a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 10 and 11 in combination.

In the present invention, it should be note that the phrase “performinga gene amplification treatment by simultaneously using oligonucleotides”means that, for example, a gene amplification treatment step asmentioned above is performed by mixing two or more pairs ofoligonucleotides and adding the obtained mixture to one reaction systemas primers. When two pairs of oligonucleotides are simultaneously used,the fungi belonging to the genus Talaromyces can be rapidly andexhaustively detected. The mixing ratio of the two pairs ofoligonucleotides is not particularly limited but is preferably 1:1 to1:2.

In the detection method of the present invention, it is preferred tosimultaneously use a detection method using the pair of theoligonucleotides (l) and (m) and/or the pair of the oligonucleotides and(n) and (o) and a detection method using the pair of theoligonucleotides (v) and (w). As mentioned above, the detection methodusing the pair of the oligonucleotides (l) and (m) or the pair of theoligonucleotides (n) and (o) can detect the fungi belonging to the genusNeosartorya and Aspergillus fumigatus, and the detection method usingthe pair of the oligonucleotides (v) and (w) can detect Aspergillusfumigatus. Therefore, when the methods are used in combination, thefungus detected from a sample can be discriminated as a fungus belongingto the genus Neosartorya or Aspergillus fumigatus. In particular, it ismore preferred to use a detection method using the pair of theoligonucleotides (l1) and (m1) and/or the pair of the oligonucleotides(n1) and (o1) and a detection method using the pair of theoligonucleotides (v1) and (w1) in combination, and it is still morepreferred to use a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 12 and 13 and/or the pair of the oligonucleotidesrepresented by the nucleotide sequences set forth in SEQ ID NOS: 14 and15 and a detection method using the pair of the oligonucleotidesrepresented by the nucleotide sequences set forth in SEQ ID NOS: 22 and23 in combination.

In view of detection sensitivity, it is preferred to use a detectionmethod using the pair of oligonucleotides (n) and (o) and a detectionmethod using the pair of oligonucleotides (v) and (w) in combination, itis more preferred to use a detection method using the pair ofoligonucleotides (n1) and (o1) and a detection method using the pair ofoligonucleotides (v1) and (w1) in combination, and it is still morepreferred to use a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 14 and 15 and a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 22 and 23 in combination.

In the detection method of the present invention, it is preferred tosimultaneously use a detection method using the pair of theoligonucleotides (p) and (q) and a detection method using the pair ofthe oligonucleotides (r) and (s) or the pair of the oligonucleotides (t)and (u). By using the methods in combination, it is possible tospecifically detect only a fungus belonging to the genus Hamigera from asample. In other words, by using the methods in combination, it ispossible to discriminate the fungus belonging to the genus Hamigera fromthe fungus belonging to the genus Cladosporium in a sample.Discrimination between the both genera enables to predict the risk ofmycotoxin, because the fungi belonging to the genus Hamigera produce nomycotoxin. In addition, it is also possible to predict whether fungalcontamination has been caused before heating or not by discriminatingbetween the both genera, because the both genera have different heatresistances. Therefore, the above detection method can be used forfinding a cause, such as revising a sterilization step or confirmingairtightness of a container.

In particular, it is more preferred to use a detection method using thepair of the oligonucleotides (p1) and (q1) and a detection method usingthe pair of the oligonucleotides (r1) and (s1) or the pair of theoligonucleotides (t1) and (u1) in combination, and it is still morepreferred to use a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 16 and 17 and a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 18 and 19 or the pair of the oligonucleotides represented bythe nucleotide sequences set forth in SEQ ID NOS: 20 and 21 incombination.

In view of detection sensitivity, it is preferred to use a detectionmethod using the pair of oligonucleotides (p) and (q) and a detectionmethod using the pair of oligonucleotides (t) and (u) in combination, itis more preferred to use a detection method using the pair ofoligonucleotides (p1) and (q1) and a detection method using the pair ofoligonucleotides (t1) and (u1) in combination, and it is still morepreferred to use a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 16 and 17 and a detection method using the pair of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 20 and 21 in combination.

In the detection method of the present invention, a preferred embodimentincludes amplifying the whole or part of any one of the nucleic acids(A-I) to (D-II) by a loop mediated isothermal amplification method (LAMPmethod).

In the LAMP method, synthesis of a complementary strand can be performedunder isothermal condition because periodic temperature control is notrequired. Therefore, a specific fungus in a sample can be detectedeasily and rapidly by the LAMP method.

Hereinafter, the detection method of the present invention by the LAMPmethod is described in detail.

The LAMP method is a loop-mediated isothermal amplification method whichdoes not require the periodic temperature control method (WO 00/28082A1), which allows isothermal complementary strand synthesis by annealingthe 3′-end side of a primer to a nucleotide serving as a template toprepare a starting point of complementary strand synthesis, andcombining a primer that anneals to a loop formed from the aboveannealing step. In the LAMP method, at least four primers whichrecognize six nucleotide sequence regions in the nucleic acid as atemplate are required, and these primers are designed so that the 3′-endside thereof is certainly annealed to the nucleotide as a template. Byusing the primers, it is possible to act a checking mechanism based oncomplementary binding of the nucleotide sequences, repeatedly, and thento perform a sensitive and specific nucleic acid amplification reaction.

The six nucleotide sequence regions recognized by the primers arereferred to as F3, F2 and F1 in this order from the 5′-end side of thenucleotide as a template, and B3c, B2c and B1c in this order from the3′-end side thereof. Complementary sequences of F1, F2 and F3 are calledF1c, F2c and F3c, respectively. Complementary sequences of B1c, B2c andB3c are called B1, B2 and B3, respectively.

The six nucleotide sequence regions may be selected by the followingprocedure, but the present invention is not limited thereto.

First, alignment of a nucleotide sequence of a fungus of interest isperformed. The alignment may be performed by using software such asClustal X. Subsequently, based on the resultant alignment information,the above-mentioned six nucleotide sequence regions are selected usingsoftware such as Primer Explorer V4 (HP of Eiken Chemical Co., Ltd.) todesign primers for the LAMP method.

Primers to be used in the LAMP method are designed by determining theabove-mentioned six nucleotide sequence regions from a nucleotidesequence of a region for amplification (a target region), and thendesigning inner primers F and B and outer primers F and B as describedbelow.

The inner primer used in the LAMP method is an oligonucleotide having,at the 3′ end, a nucleotide sequence that recognizes a certainnucleotide sequence region in a target nucleotide sequence and providesa synthesis origin; and having, at the 5′ end, a nucleotide sequencecomplementary to an arbitrary region of a nucleic acid synthesisreaction product obtained with this primer at the origin. In the innerprimers, a primer having a “nucleotide sequence selected from F2” at the3′ end and a “nucleotide sequence selected from F1c” at the 5′ end iscalled an inner primer F (hereinafter, abbreviated to FIP), and a primerhaving a “nucleotide sequence selected from B2” at the 3′ end and a“nucleotide sequence selected from B1c” at the 5′ end is called an innerprimer B (hereinafter, abbreviated to BIP). The inner primers may havearbitrary nucleotide sequences including 0 to 50 nucleotides at aposition between the F2 region and the F1c region or between the B2region and the B1c region.

The outer primer used in the LAMP method is an oligonucleotide having anucleotide sequence that recognizes “a certain nucleotide sequenceregion which presents on the 5′-end side of a certain nucleotidesequence region such as the above-mentioned F2 region or B2 region” inthe target nucleotide sequence and provides a synthesis origin. Examplesthereof include a primer including a nucleotide sequence selected fromthe F3 region and a primer including a nucleotide sequence selected fromthe B3 region. In the outer primers, a primer containing a “nucleotidesequence selected from F3” is called an outer primer F (hereinafter,abbreviated to F3), and a primer containing a “nucleotide sequenceselected from B3” is called an outer primer B (hereinafter, abbreviatedto B3).

“F” in each primer means that a primer complementarily binds to theanti-sense strand of the target nucleotide sequence and provides asynthesis origin. “B” in each primer i means that a primercomplementarily binds to the sense strand of the target nucleotidesequence and provides a synthesis origin.

In the amplification of the nucleic acid by the LAMP method, a loopprimer(s) (hereinafter, abbreviated to LF and LB) can be preferably usedin addition to the inner and outer primers. The loop primers refer to 2primers (one for each of strands composing a double-strand) containing,at the 3′ end, a nucleotide sequence complementary to a sequence in aloop formed by the annealing of complementary sequences present at thesame strand of an amplification product obtained by the LAMP method.That is, the loop primer is a primer having a nucleotide sequencecomplementary to a nucleotide sequence of a single strand moiety of theloop structure on the 5′-end side in the dumbbell-like structure. Theuse of the loop primers increases nucleic acid synthesis origins innumber and achieves reduction in reaction time and enhancement indetection sensitivity (WO 02/24902 Pamphlet).

The nucleotide sequence of the loop primer may be selected fromnucleotide sequences in the target region or complementary strandsthereof or may be another nucleotide sequence, as long as the sequenceis complementary to the nucleotide sequence of the single strand moietyof the loop structure on the 5′-end side in the dumbbell-like structure.Further, one type or two types of loop primers may be used.

When a DNA fragment of the target region is amplified using at leastfour or more types of the above-mentioned primers, the DNA fragment canbe amplified to an amount sufficient for specific and efficientdetection of the DNA fragment. Therefore, a specific fungus can bedetected by confirming whether the amplified product is present or not.

In the method of the present invention, when detecting theheat-resistant fungi by the RAMP method, the oligonucleotide fordetecting is preferably an oligonucleotide consisting of a nucleotidesequence corresponding to any one of the following (i) to (vi). In thefollowing (i) to (vi), nucleotide sequence regions F3, F2 and F1 areselected from the 5′-end side in a target region selected from thenucleotide sequence of the variable region in the β-tubulin gene or theD1/D2 region and ITS region of 28S rDNA of the heat-resistant fungus,nucleotide sequence regions B3c, B2c and B1c are selected from the3′-end side in the target region, complementary nucleotide sequences ofthe B3c, B2c and B1c are called B3, B2 and B1, respectively, andcomplementary nucleotide sequences of the F3, F2 and F1 are called F3c,F2c and F1c, respectively.

(i) A nucleotide sequence having the sequence identical to that of theB2 region at the 3′ terminal side and the sequence identical to that ofthe B1c region at the 5′ terminal side

(ii) A nucleotide sequence having the sequence identical to that of theB3 region

(iii) A nucleotide sequence having the sequence identical to that of theF2 region at the 3′ terminal side and the sequence identical to that ofthe F1c region at the 5′ terminal side

(iv) A nucleotide sequence having the sequence identical to that of theF3 region

(v) A nucleotide sequence having a sequence complementary to a partbetween the B1 region and the B2 region

(vi) A nucleotide sequence having a sequence complementary to a partbetween the F1 region and the F2 region

The oligonucleotide may be used not only as a primer for the LAMP methodbut also as, for example, a primer for the PCR method or a probe fordetecting a nucleic acid.

The primer that can be used in the present invention preferably includes15 or more nucleotides, and more preferably 20 or more nucleotides.Further, each primer may be an oligonucleotide of single nucleotidesequence or a mixture of oligonucleotides of a plurality of nucleotidesequences.

In the present invention, in the case where the heat-resistant fungus isdetected by the LAMP method, the variable region in the β-tubulin geneor the D1/D2 region and ITS region of 28S rDNA of the heat-resistantfungus (i.e., the whole or part of any one of the nucleic acids (A-I) to(D-II)) is determined as a target region, and a DNA fragment includingthe region is amplified to confirm whether an amplification product ispresent or not.

A region to be amplified by the LAMP method preferably includes; anucleotide sequence which is part of the nucleotide sequence set forthin SEQ ID NO: 25 and includes the whole or part of the nucleotidesequence of position 400 to 600 in the nucleotide sequence set forth inSEQ ID NO: 25; a nucleotide sequence which is part of the nucleotidesequence set forth in SEQ ID NO: 32 and includes the whole or part ofthe nucleotide sequence of position 10 to 250 and/or position 350 to 559in the nucleotide sequence set forth in SEQ ID NO: 32; a nucleotidesequence which is part of the nucleotide sequence set forth in SEQ IDNO: 34 and includes the whole or part of the nucleotide sequence ofposition 10 to 250 and/or position 350 to 559 in the nucleotide sequenceset forth in SEQ ID NO: 34; a nucleotide sequence which is part of thenucleotide sequence set forth in SEQ ID NO: 35 and includes the whole orpart of the nucleotide sequence of position 300 to 550 in the nucleotidesequence set forth in SEQ ID NO: 35; a nucleotide sequence which is partof the nucleotide sequence set forth in SEQ ID NO: 26 and includes thewhole or part of the nucleotide sequence of position 200 to 450 in thenucleotide sequence set forth in SEQ ID NO: 26; a nucleotide sequencewhich is part of the nucleotide sequence set forth in SEQ ID NO: 29 andincludes the whole or part of the nucleotide sequence of position 150 to420 in the nucleotide sequence set forth in SEQ ID NO: 29; a nucleotidesequence which is part of the nucleotide sequence set forth in SEQ IDNO: 27 and includes the whole or part of the nucleotide sequence ofposition 150 to 450 in the nucleotide sequence set forth in SEQ ID NO:27; a nucleotide sequence which is part of the nucleotide sequence setforth in SEQ ID NO: 30 and includes the whole or part of the nucleotidesequence of position 250 to 550 in the nucleotide sequence set forth inSEQ ID NO: 30; a nucleotide sequence which is part of the nucleotidesequence set forth in SEQ ID NO: 31 and includes the whole or part ofthe nucleotide sequence of position 250 to 550 in the nucleotidesequence set forth in SEQ ID NO: 31; or a nucleotide sequence resultingfrom a deletion, substitution, or addition of one to several nucleotidesin any one of the above-mentioned nucleotide sequences.

Hereinafter, primers and primer sets which are preferably used indetection of the heat-resistant fungi by the LAMP method are described.

In order to design primers for specifically detecting the fungibelonging to the genus Byssochlamys, the above-mentioned six nucleotidesequence regions are preferably determined from the range in thenucleotide numbers 400 to 600 of the nucleotide sequence set forth inSEQ ID NO: 25. Specifically, it is preferred to use a primer consistingof a oligonucleotide having the nucleotide sequence set forth in SEQ IDNO: 36, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 37, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:38, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 39, and a primer set including theprimers, and it is more preferred to use the following primer set.

Primer Set for Detecting the Fungi Belonging to the Genus Byssochlamys(LB1 Primer Set)

LB1F3 primer: (SEQ ID NO: 36) CGGTCCTCGAGCGTATGG LB1B3 primer:(SEQ ID NO: 37) CCGTTACTGGGGCAATCC LB1FIP primer: (SEQ ID NO: 38)AGTTAGGTGACCGTGAGGTCGTCTTTGTCACGCGCTCTGG LB1BIP primer: (SEQ ID NO: 39)GGATCAGGTAGGGATACCCGCTGTTGGTTTCTTTTCCTCCGC

FIG. 26 illustrates positions of nucleotide sequences recognized by theabove-mentioned primers in the nucleotide sequence of the ITS region andD1/D2 region of 28S rDNA of the fungi belonging to the genusByssochlamys.

When the primers and primer set are used, the variable region in the ITSregion and D1/D2 region of 28S rDNA of the fungi belonging to the genusByssochlamys can be amplified specifically, rapidly, and sensitively bythe LAMP method. Therefore, the fungi belonging to the genusByssochlamys in a sample can be detected by confirming amplification ofthe DNA fragments.

In order to design primers for specifically detecting the fungibelonging to the genus Neosartorya and Aspergillus fumigatus, theabove-mentioned six nucleotide sequence regions are preferablydetermined from the range in the nucleotide numbers 350 to 559 and therange in the nucleotide numbers 10 to 250 of the nucleotide sequence setforth in SEQ ID NO: 32 or 34. Specifically, it is preferred to use aprimer consisting of a oligonucleotide having the nucleotide sequenceset forth in SEQ ID NO: 40, a primer consisting of a oligonucleotidehaving the nucleotide sequence set forth in SEQ ID NO: 41, a primerconsisting of a oligonucleotide having the nucleotide sequence set forthin SEQ ID NO: 42, a primer consisting of a oligonucleotide having thenucleotide sequence set forth in SEQ ID NO: 43, and a primer setincluding the primers, and it is more preferred to use the followingprimer set.

Primer Set for Detecting the Fungi Belonging to the Genus Neosartoryaand Aspergillus fumigatus (LN1 Primer Set)

LN1F3 primer: (SEQ ID NO: 40) GGCAACATCTCACGATCTGA LN1B3 primer:(SEQ ID NO: 41) CCCTCAGTGTAGTGACCCTT LN1FIP primer: (SEQ ID NO: 42)ATGGTACCAGGCTCGAGATCGATACTAGGCCAACGGTGACA LN1BIP primer: (SEQ ID NO: 43)GTCCCTTCGGCGAGCTCTTCGTTGTTACCAGCACCAGACT

To detect the fungi belonging to the genus Neosartorya and Aspergillusfumigatus, loop primers are preferably used in addition to theabove-mentioned primers. The following primers are preferably used asthe loop primers. Moreover, the primer set preferably further includesprimers consisting of oligonucleotides having the nucleotide sequencesset forth in SEQ ID NOS: 44 and 45.

Loop Primer for Detecting the Fungi Belonging to the Genus Neosartoryaand Aspergillus fumigatus (LN1 Loop Primer)

(SEQ ID NO: 44) LN1LF loop primer: ACGGCACGAGGAACATACT (SEQ ID NO: 45)LN1LB loop primer: CGATAACTTCGTCTTCGGCC

FIG. 27 illustrates positions of nucleotide sequences recognized by theabove-mentioned primers in the nucleotide sequence of the β-tubulin geneof Neosartorya fischeri which belongs to the genus Neosartorya includingNeosartorya glabra.

When the primers and primer set are used, the variable region in theβ-tubulin gene of the fungi belonging to the genus Neosartorya andAspergillus fumigatus can be amplified specifically, rapidly, andsensitively by the LAMP method. Therefore, the fungi belonging to thegenus Neosartorya and/or Aspergillus fumigatus in a sample can bedetected by confirming amplification of the DNA fragments.

In order to design primers for specifically detecting Aspergillusfumigatus, other than the above-mentioned primer set, it is preferred touse a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 46, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:47, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 48, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:49, and a primer set including the primers, and it is more preferred touse the following primer set.

Primer Set for Detecting Aspergillus fumigatus (LAf2 Primer Set)

LAf2F3 primer: (SEQ ID NO: 46) GCCGCTTTCTGGTATGTCT LAf2B3 primer:(SEQ ID NO: 47) CGCTTCTTCCTTGTTTTCCG LAf2FIP primer: (SEQ ID NO: 48)CCATGACAGTGAGGCTGAACCCCGGGTGATTGGGATCTCTCA LAf2BIP primer:(SEQ ID NO: 49) ACCATCTCTGGTGAGCATGGCTTTCCGCCGCTTTCTCAA

To detect the fungi belonging to Aspergillus fumigatus, loop primers arepreferably used in addition to the above-mentioned primers. Thefollowing primer is preferably used as the loop primer. Moreover, theprimer set preferably further includes a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:50.

Loop Primer for Detecting Aspergillus fumigatus (LAf2 Loop Primer)

(SEQ ID NO: 50) LAf2LB loop primer: AGTAAGTTCGACCTATATCCTCCC

FIG. 28 illustrates positions of nucleotide sequences recognized by theabove-mentioned primers in the nucleotide sequence of the β-tubulin geneof Aspergillus fumigatus. When the primers and primer set are used, thevariable region in the β-tubulin gene of Aspergillus fumigatus can beamplified specifically, rapidly, and sensitively by the LAMP method.Therefore, Aspergillus fumigatus in a sample can be detected byconfirming amplification of the DNA fragments.

It should be noted that, when the primer set is used, it is possible tospecifically detect Aspergillus fumigatus but is impossible to detectthe fungi belonging to the genus Neosartorya. Therefore, the fungibelonging to the genus Neosartorya can be discriminated from Aspergillusfumigatus by using the primer set for detecting the fungi belonging tothe genus Neosartorya and Aspergillus fumigatus (LN1 primer set) and theprimer set for detecting Aspergillus fumigatus (LAf2 primer set) incombination.

In order to design primers for specifically detecting the fungibelonging to the genus Hamigera, the above-mentioned six nucleotidesequence regions are preferably determined from the range in thenucleotide numbers 300 to 550 of the nucleotide sequence set forth inSEQ ID NO: 35. Specifically, it is preferred to use a primer consistingof a oligonucleotide having the nucleotide sequence set forth in SEQ IDNO: 51, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 52, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:53, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 54, and a primer set including theprimers, and it is more preferred to use the following primer set.

Primer Set for Detecting the Fungi Belonging to the Genus Hamigera (LH2Primer Set)

LH2F3 primer: (SEQ ID NO: 51) GGATCCGAATACGACGTGTC LH2B3 primer:(SEQ ID NO: 52) CCCTCAGTGTAGTGACCCTT LH2FIP primer: (SEQ ID NO: 53)CATGGTGCCAGGCTCGAGATCCAGGCCAGCGGTAACAAG LH2BIP primer: (SEQ ID NO: 54)CCGGTCCTTTTGGCCAGCTCTGTTACCGGCACCAGACT

To detect the fungi belonging to the genus Hamigera, loop primers arepreferably used in addition to the above-mentioned primers. Thefollowing primers are preferably used as the loop primers. Moreover, theprimer set preferably further includes primers consisting ofoligonucleotides having the nucleotide sequences set forth in SEQ IDNOS: 55 and 56.

Loop Primer for Detecting the Fungi Belonging to the Genus Hamigera (LH2Loop Primer)

(SEQ ID NO: 55) LH2LF loop primer: ACGGCACGGGGGACATA (SEQ ID NO: 56)LH2LB loop primer: TTCCGCCCAGACAACTTCG

FIG. 29 illustrates positions of nucleotide sequences recognized by theabove-mentioned primers in the nucleotide sequence of the β-tubulin geneof the fungi belonging to the genus Hamigera.

When the primers and primer set are used, the variable region in theβ-tubulin gene of the fungi belonging to the genus Hamigera can beamplified specifically, rapidly, and sensitively by the LAMP method.Therefore, the fungi belonging to the genus Hamigera in a sample can bedetected by confirming amplification of the DNA fragments.

In order to design primers for specifically detecting Talaromycesflavus, the above-mentioned six nucleotide sequence regions arepreferably determined from the range in the nucleotide numbers 200 to450 of the nucleotide sequence set forth in SEQ ID NO: 26. Specifically,it is preferred to use a primer consisting of a oligonucleotide havingthe nucleotide sequence set forth in SEQ ID NO: 57, a primer consistingof a oligonucleotide having the nucleotide sequence set forth in SEQ IDNO: 58, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 59, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:60, and a primer set including the primers, and it is more preferred touse the following primer set.

Primer Set for Detecting Talaromyces flavus (LTf2 Primer Set)

LTf2F3 primer: (SEQ ID NO: 57) CCAGTTGGAGCGTATGAACG LTf2B3 primer:(SEQ ID NO: 58) CCCAGTTGTTACCAGCACCG LTf2FIP primer: (SEQ ID NO: 59)TTGTTGCCGGAGGCCTACACTTTACTTCAACGAGGTGCGT LTf2BIP primer: (SEQ ID NO: 60)CGACTTGGAGCCCGGTACCAAAAGTTGTCGGGACGGAAGA

To detect Talaromyces flavus, loop primers are preferably used inaddition to the above-mentioned primers. The following primer ispreferably used as the loop primer. Moreover, the primer set preferablyfurther includes a primer consisting of a oligonucleotide having thenucleotide sequence set forth in SEQ ID NO: 61.

Loop Primer for Detecting Talaromyces flavus (LTf2 Loop Primer)

(SEQ ID NO: 61) LTf2LB loop primer: GCTGGTCCCTTTGGTCAGC

FIG. 30 illustrates positions of nucleotide sequences recognized by theabove-mentioned primers in the nucleotide sequence of the β-tubulin geneof Talaromyces flavus. When the primers and primer set are used, thevariable region in the β-tubulin gene of Talaromyces flavus can beamplified specifically, rapidly, and sensitively by the LAMP method.Therefore, Talaromyces flavus in a sample can be detected by confirmingamplification of the DNA fragments.

In order to design primers for specifically detecting Talaromyceswortmannii, the above-mentioned six nucleotide sequence regions arepreferably determined from the range in the nucleotide numbers 150 to420 of the nucleotide sequence set forth in SEQ ID NO: 29. Specifically,it is preferred to use a primer consisting of a oligonucleotide havingthe nucleotide sequence set forth in SEQ ID NO: 62, a primer consistingof a oligonucleotide having the nucleotide sequence set forth in SEQ IDNO: 63, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 64, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:65, and a primer set including the primers, and it is more preferred touse the following primer set.

Primer Set for Detecting Talaromyces wortmannii (LTw4-3 Primer Set)

LTw4F3 primer: (SEQ ID NO: 62) TGGCTCCGGAATGTGAGTT LTw3B3 primer:(SEQ ID NO: 63) CAAATCGACGAGGACGGC LTw4FIP primer: (SEQ ID NO: 64)CGCTCCAACTGGAGGTCGGAAAATTTCGACATCCCACCCT LTw3BIP primer: (SEQ ID NO: 65)GGAATCTGCCCCGCGACATTCCGGGGGACGTACTTGTTG

To detect Talaromyces wortmannii, loop primers are preferably used inaddition to the above-mentioned primers. The following primers arepreferably used as the loop primers. Moreover, the primer set preferablyfurther includes primers consisting of oligonucleotides having thenucleotide sequences set forth in SEQ ID NOS: 66 and 67.

Loop Primer for Detecting Talaromyces wortmannii (LTw4-3 Loop Primer)

(SEQ ID NO: 66) LTw4LF loop primer: GGTGCCATTGTAACTGGAAATGA(SEQ ID NO: 67) LTw3LB loop primer: ACTCATATCGTATAGGCTAGCGG

FIG. 31 illustrates positions of nucleotide sequences recognized by theabove-mentioned primers in the nucleotide sequence of the β-tubulin geneof Talaromyces wortmannii.

When the primers and primer set are used, the variable region in theβ-tubulin gene of Talaromyces wortmannii can be amplified specifically,rapidly, and sensitively by the LAMP method. Therefore, Talaromyceswortmannii in a sample can be detected by confirming amplification ofthe DNA fragments.

In order to design primers for specifically detecting Talaromycesluteus, the above-mentioned six nucleotide sequence regions arepreferably determined from the range in the nucleotide numbers 150 to450 of the nucleotide sequence set forth in SEQ ID NO: 27. Specifically,it is preferred to use a primer consisting of a oligonucleotide havingthe nucleotide sequence set forth in SEQ ID NO: 68, a primer consistingof a oligonucleotide having the nucleotide sequence set forth in SEQ IDNO: 69, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 70, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:71, and a primer set including the primers, and it is more preferred touse the following primer set.

Primer Set for Detecting Talaromyces luteus (LTI1 Primer Set)

LTI1F3 primer: (SEQ ID NO: 68) CGAATCACCACTGATGGGAA LTI1B3 primer:(SEQ ID NO: 69) GAAGAGCTGACCGAAAGGAC LTI1FIP primer: (SEQ ID NO: 70)TTCGTGCTGTCGGTCGGTAATGTTCCGACCTCCAGTTAGAGC LTI1BIP primer:(SEQ ID NO: 71) TAGGCTAGCGGCAACAAGTACGATAGTACCGGGCTCCAGATC

To detect Talaromyces luteus, loop primers are preferably used inaddition to the above-mentioned primers. The following primer ispreferably used as the loop primer. Moreover, the primer set preferablyfurther includes a primer consisting of a oligonucleotide having thenucleotide sequence set forth in SEQ ID NO: 72.

Loop Primer for Detecting Talaromyces luteus (LTI1 Loop Primer)

(SEQ ID NO: 72) LTI1LF loop primer: ACCTCGTTGAAATAGACGTTCA

FIG. 32 illustrates positions of nucleotide sequences recognized by theabove-mentioned primers in the nucleotide sequence of the β-tubulin geneof Talaromyces luteus. When the primers and primer set are used, thevariable region in the β-tubulin gene of Talaromyces luteus can beamplified specifically, rapidly, and sensitively by the LAMP method.Therefore, Talaromyces luteus in a sample can be detected by confirmingamplification of the DNA fragments.

In order to design primers for specifically detecting Talaromyces flavusand Talaromyces trachyspermus, the above-mentioned six nucleotidesequence regions are preferably determined from the range in thenucleotide numbers 250 to 550 of each nucleotide sequence set forth inSEQ ID NO: 30 or 31. Specifically, it is preferred to use a primerconsisting of a oligonucleotide having the nucleotide sequence set forthin SEQ ID NO: 73, a primer consisting of a oligonucleotide having thenucleotide sequence set forth in SEQ ID NO: 74, a primer consisting of aoligonucleotide having the nucleotide sequence set forth in SEQ ID NO:75, a primer consisting of a oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 76, and a primer set including theprimers, and it is more preferred to use the following primer set.

Primer Set for Detecting Talaromyces flavus and TalaromycesTrachyspermus (LT1 Primer Set)

LT1F3 primer: (SEQ ID NO: 73) GCGTCATTTCTGCCCTCAA LT1B3 primer:(SEQ ID NO: 74) AGTTCAGCGGGTAACTCCT LT1FIP primer: (SEQ ID NO: 75)TACGCTCGAGGACCAGACGGCGGCTTGTGTGTTGGGTG LT1BIP primer: (SEQ ID NO: 76)TCTGTCACTCGCTCGGGAAGGACCTGATCCGAGGTCAACC

To detect Talaromyces flavus and Talaromyces trachyspermus, loop primersare preferably used in addition to the above-mentioned primers. Thefollowing primers are preferably used as the loop primers. Moreover, theprimer set preferably further includes primers consisting of theoligonucleotides having the nucleotide sequences set forth in SEQ IDNOS: 77 and 78.

Loop Primer for Detecting Talaromyces flavus and TalaromycesTrachyspermus (LT1 Loop Primer)

(SEQ ID NO: 77) LT1LF loop primer: GCTGCCTTTTGGGCAGGTC (SEQ ID NO: 78)LT1LB loop primer: TGGTCACACCACTATATTTTACCAC

FIG. 33, FIG. 34 and FIG. 34-1 illustrate positions of nucleotidesequences recognized by the above-mentioned primers in the nucleotidesequence of the ITS region and D1/D2 region of 28S rDNA of Talaromycesflavus and Talaromyces trachyspermus.

When the primers and primer set are used, the variable region in the ITSregion and D1/D2 region of 28S rDNA of Talaromyces flavus andTalaromyces trachyspermus can be amplified specifically, rapidly, andsensitively by the LAMP method. Therefore, Talaromyces flavus andTalaromyces trachyspermus in a sample can be detected by confirmingamplification of the DNA fragments.

The outer primers in the above-mentioned primer sets may be used notonly in the LAMP method but also in the PCR method. In the PCR method, aDNA fragment of interest can be amplified by PCR using theabove-mentioned primers, the β-tubulin gene or the ITS region and D1/D2region of 28S rDNA in a sample as a template, and a heat-stable DNApolymerase.

An enzyme used in amplification of the DNA fragment is not particularlylimited as long as it is generally used, and it is preferably atemplate-dependent nucleic acid synthetase having strand displacementactivities. Such an enzyme includes Bst DNA polymerase (large fragment),Bca (exo-) DNA polymerase, and the Klenow fragment of E. coli DNApolymerase I; and preferably includes Bst DNA polymerase (largefragment). The enzyme that can be used in the present invention may bepurified from viruses, bacteria, or the like or may be prepared by agene recombination technique. These enzymes may be modified byfragmentation, amino acid substitution, or the like.

The temperature for amplification by the LAMP method is not particularlylimited but is preferably 60 to 65° C.

The amplification of the DNA fragment by the LAMP method can beconfirmed by general method. The nucleic acid amplification products canbe detected, for example, by hybridization using a labeledoligonucleotide as a probe which specifically recognizes amplifiednucleotide sequences; by a fluorescent intercalator method(JP-A-2001-242169); by directly applying the reaction solution after thecompletion of reaction to agarose gel electrophoresis. In case of theagarose gel electrophoresis, the LAMP amplification products aredetected in the form of a ladder of many bands differing in base length.

Moreover, in the LAMP method, substrates are consumed in large amountsby nucleic acid synthesis, and pyrophosphoric acid ions as by-productsare converted into magnesium pyrophosphate through its reaction withcoexisting magnesium ions and makes the reaction solution cloudy to theextent that can be observed visually. Thus, the nucleic acidamplification reaction may be detected by confirming this cloudiness byuse of a measurement apparatus that can optically observe time-dependentrises in turbidity after the completion of reaction or during reaction,for example, by confirming changes in absorbance at 400 nm by use of aspectrophotometer (WO 01/83817 Pamphlet).

According to the detection method by the LAMP method, a procedure from asample preparation step to a fungus detection step can be performedwithin a time as short as about 60 to 120 minutes.

Hereinafter, an embodiment of the method of detecting the heat-resistantfungus of the present invention will be described specifically, but thepresent invention is not limited thereto.

1) Analysis of Fungus Causing Accident

Foods and drinks which contain sugars and proteins and cannot besterilized under strong conditions may cause contamination accidents byheat-resistant fungi. Examples of the foods and drinks include foods anddrinks made from agricultural products such as fruits and fruit juicesand animal products such as milk.

In the detection method, DNA is collected from hyphae detected from adrink or the like which has caused the accident. Thereafter, a geneamplification treatment such as PCR reactions or the LAMP method isperformed using the above-mentioned oligonucleotides of the presentinvention as nucleic acid primers.

In the case where the gene amplification is performed by the PCR method,the pair of the oligonucleotides (a) and (b) is used as a primer pairfor detecting the genus Byssochlamys; one or more pairs of theoligonucleotides (c) and (d), the pair of the oligonucleotides (e) and(f), the pair of the oligonucleotides (g) and (h), the pair of theoligonucleotides (i) and (h), and the pair of the oligonucleotides (j)and (k) are used as primers for detecting the genus Talaromyces; one ormore pairs of the oligonucleotides (l) and (m) and the pair of theoligonucleotides (n) and (o) are used as primers for detecting the genusNeosartorya and Aspergillus fumigatus; and one or more pairs of theoligonucleotides (p) and (q), the pair of the oligonucleotides (r) and(s), and the pair of the oligonucleotides (t) and (u) are used asprimers for detecting the genus Hamigera.

In the case where the gene amplification is performed by the LAMPmethod, the set of the oligonucleotide represented by the nucleotidesequences set forth in SEQ ID NOS: 36 to 39 is used as primers fordetecting the genus Byssochlamys; one or more sets of the set of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 57 to 61, the set of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 62 to 67, the set of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 68 to 72, and the set of the oligonucleotides represented bythe nucleotide sequences set forth in SEQ ID NOS: 73 to 78 are used asprimers for detecting the genus Talaromyces; the set of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 40 to 45 is used as primers for detecting fungi belonging tothe genus Neosartorya and Aspergillus fumigatus; and the set of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 51 to 56 is used as primers for detecting fungi belonging tothe genus Hamigera.

When the primer pairs and primer sets are used, the above-mentioned fourgenera of heat-resistant fungi can be independently detected. Moreover,when the primers for detecting the respective genera are used inappropriate combination, a plurality of genera and species in the fourgenera can be exhaustively detected. The primers of the presentinvention are specific to each genus, and hence it is possible toexhaustively detect the four genera of heat-resistant fungi and toidentify fungi based on the type of primers used at genus level(detection of heat-resistant fungi in the four genera).

After the gene amplification treatment using the primers, in the case ofthe PCR method, electrophoresis is performed to confirm whether theamplification product is present or not, while in the case of the LAMPmethod, the turbidity of the reaction solution is measured to confirmwhether the amplification reaction is caused or not. At the same time,part of the fungal cells collected is inoculated intochloramphenicol-supplemented PDA and cultured at 50° C.

In the case where a PCR gene amplification product is confirmed by usingprimers other than the primers for detecting fungi belonging to thegenus Neosartorya, and/or in the case where a gene amplification productis confirmed by using the primers for detecting fungi belonging to thegenus Neosartorya and hyphae do not elongate at 50° C., or no reactionproduct is detected after PCR reactions or LAMP reactions using theprimers for discriminating Aspergillus fumigatus from Neosartorya, thesample is evaluated to be “positive for heat-resistant fungi (includingfungi belonging to the genus Neosartorya)”.

As an example, a case where a gene amplification treatment is performedby the PCR method using the pair of the oligonucleotides (a) and (b),the pair of the oligonucleotides (t) and (u), the pair of theoligonucleotides (n) and (o), the pair of the oligonucleotides (i) and(h), and the pair of the oligonucleotides (j) and (k) in combination isdescribed in detail. In the case where a reaction product is confirmedin a system using the primers for detecting fungi belonging to the genusNeosartorya (the pair of the oligonucleotides (n) and (o)), elongationof hyphae cultured at 50° C. is confirmed to discriminate that thefungus is Neosartorya or Aspergillus fumigatus. Alternatively, detectionis performed using the oligonucleotides for Neosartorya and Aspergillusfumigatus as nucleic acid primers. For example, PCR reactions areperformed using the oligonucleotides (v) and (w) as primers, in the casewhere a reaction product of about 200 bp is detected, the sample isevaluated to be positive for Aspergillus fumigatus, while in the casewhere no reaction product is detected, the sample is evaluated to bepositive for fungi belonging to the genus Neosartorya.

In the case where a reaction product is confirmed in a system using theprimers for detecting fungi belonging to the genus Byssochlamys (thepair of the oligonucleotides (a) and (b)), the sample is evaluated to bepositive for fungi belonging to the genus Byssochlamys.

In the case where a reaction product is confirmed in a system using theprimers for detecting fungi belonging to the genus Hamigera (the pair ofthe oligonucleotides (t) and (u)), the sample is evaluated to bepositive for fungi belonging to the genus Hamigera.

In the case where a reaction product is confirmed in a system using theprimers for detecting fungi belonging to the genus Talaromyces (the pairof the oligonucleotides (j) and (k) and/or the pair of theoligonucleotides (i) and (h)), the sample is evaluated to be positivefor fungi belonging to the genus Talaromyces.

The heat-resistant fungi survive under heat sterilization conditions,and hence it is highly likely that the heat-resistant fungi were mixedin the sample from raw materials and steps. Therefore, in the case wherethe sample is positive for the heat-resistant fungi, it is necessary tofurther test the cleanliness level of the raw materials and productionenvironment. On the other hand, in the case where the sample is negativefor the heat-resistant fungi, it is necessary to revise thesterilization step or to confirm airtightness of a container because thecontamination was caused by a fault in sterilization or mixing of thefungi in the sample after sterilization (investigation into the cause).

2) Microorganism Inspection of Raw Material

A usual fungus inspection of raw materials requires two tests forgeneral fungi (a sample is not subjected to a heat shock treatment) andfor heat-resistant fungi (a sample is subjected to a heat shocktreatment). In contrast, it is not necessary to perform the heat shocktreatment for the sample according to the present invention. That is,the test for general fungi is only performed, and then, in the casewhere hyphae are confirmed, the hyphae may be used to evaluate whetherthe fungi are heat-resistant fungi or not by the method of 1) above. Ina conventional method, it takes about seven days to detectheat-resistant fungi. In contrast, according to the present invention,it is possible to reduce the time for detection by two days because ittakes about three days to confirm the hyphae and at most two days todetect the fungi. Moreover, in the conventional method, it further takesabout seven days to identify the species of the fungi after detection ofthe heat-resistant fungi, while in the method of the present invention,it is possible to simultaneously perform detection and identification atgenus level.

The sample to be used for the detection method of the present inventionis not particularly limited, and may be a food or drink itself, a rawmaterial of the food or drink, an isolated fungus, a cultured fungus, orthe like.

A method of preparing DNA from a sample is not particularly limited aslong as DNA can be obtained at a sufficient purification degree and in asufficient amount for detecting the heat-resistant fungi. DNA obtainedby reverse transcriptase from RNA contained in a sample may be used.While the sample may be used without purification, the sample may besubjected to a pre-treatment such as separation, extraction,concentration, or purification before use. For example, the sample maybe purified by phenol and chloroform extraction or using a commerciallyavailable extraction kit to increase the purity of the nucleic acidbefore use.

According to the method of the present invention, a procedure from asample preparation step to a fungus detection step can be performedwithin a time as short as about 5 to 12 hours.

The kit for detecting a heat-resistant fungus of the present inventionincludes the above-mentioned oligonucleotides for detection as a nucleicacid probe or a nucleic acid primer. Specifically, the kit for detectinga heat-resistant fungus of the present invention includes, as a nucleicacid probe or a nucleic acid primer, at least one oligonucleotideselected from the group consisting of oligonucleotides which canhybridize with the nucleic acid (I) or (II) and can act asoligonucleotides for specifically detecting the heat-resistant fungus.The kit particularly preferably includes, as a nucleic acid probe or anucleic acid primer, at least one oligonucleotide selected from thegroup consisting of an oligonucleotide including the nucleotide sequenceset forth in any one of SEQ ID NOS: 1 to 23 or 36 to 78 or acomplementary sequence thereof and an oligonucleotide including anucleotide sequence which has 70% or more homology to the nucleotidesequence or the complementary sequence thereof and can act as anoligonucleotide for detection. The kit can be used for detection of theheat-resistant fungi. The kit of the present invention may include notonly the above-mentioned nucleic acid probes or nucleic acid primers butalso, depending on purpose, substances which are usually used fordetecting a fungus, such as a label-detecting substance, a buffer, anucleic acid synthetase (such as a DNA polymerase, an RNA polymerase, ora reverse transcriptase), and an enzyme substrate (such as dNTP orrNTP).

For example, the kit for detecting the heat-resistant fungus by LAMPmethod preferably includes the above-mentioned primer set, a variety ofoligonucleotide(s) necessary as a loop primer, four types of dNTPsserving as substrates of nucleic acid synthesis (dATP, dCTP, dGTP, anddTTP), a DNA polymerase such as a template-dependent nucleic acidsynthetase having strand displacement activity, a buffer which providespreferred conditions for enzymatic reactions, a salt serving as acofactor (such as a magnesium salt or a manganese salt), and aprotecting agent for stabilizing an enzyme or a template, and ifnecessary, reagents necessary for detection of reaction products. Thekit of the present invention may include a positive control forconfirming whether gene amplification proceeds normally by theoligonucleotides for detection of the present invention. The positivecontrol is, for example, DNA including a region which is amplified bythe oligonucleotides for detection of the present invention.

EXAMPLES

Hereinafter, the present invention will be described more in detail withreference to Examples, but it should be understood that thetechnological scope of the present invention is not particularly limitedby the following Examples.

Example 1 (A-1) Detection and Discrimination of Fungi Belonging to theGenus Byssochlamys

1. Determination of Partial Nucleotide Sequence of β-Tubulin Gene

Nucleotide sequences of the β-tubulin gene of each variety of fungibelonging to the genus Byssochlamys were determined by the followingmethod.

A test fungus was cultured in the dark on a potato dextrose agar slantat 30° C. for 7 days. DNA was extracted from the fungus usingGenTorukun™ (manufactured by TAKARA BIO INC.). PCR amplification of atarget site was performed using PuRe Taq™ Ready-To-Go PCR Beads(manufactured by GE Health Care UK LTD); and primers Bt2a(5′-GGTAACCAAATCGGTGCTGCTTTC-3′, SEQ ID NO: 79) and Bt2b(5′-ACCCTCAGTGTAGTGACCCTTGGC-3′, SEQ ID NO: 80) (Glass and Donaldson,Appl Environ Microbiol 61: 1323-1330, 1995). Amplification of β-tubulinpartial length was performed under conditions including a denaturationtemperature of 95° C., an annealing temperature of 59° C., an elongationtemperature of 72° C., and 35 cycles. PCR products were purified usingAuto Seg™ G-50 (manufactured by Amersham Pharmacia Biotech). The PCRproducts were labeled with Big Dye (registered trademark) terminatorVer. 1.1 (manufactured by Applied Biosystems), and electrophoresis wasperformed using ABI PRISM 3130 Genetic Analyzer (manufactured by AppliedBiosystems). Nucleotide sequences from fluorescence signals inelectrophoresis were determined using the software “ATGC Ver. 4”(manufactured by Genetyx).

Based on the nucleotide sequence information of the β-tubulin gene ofByssochlamys nivea and known nucleotide sequence information of theβ-tubulin gene of a variety of fungi, alignment analyses were performedusing DNA analysis software (product name: DNAsis pro, manufactured byHitachi Software Engineering Co., Ltd.), to thereby determine a specificregion in the β-tubulin gene including nucleotide sequences specific tothe fungi belonging to the genus Byssochlamys (SEQ ID NO: 24).

2. Detection of Fungi Belonging to the Genus Byssochlamys andIdentification of the Fungi at Genus Level

(1) Design of Primers

From regions having particularly high specificity to the fungi belongingto the genus Byssochlamys on the 3′-end side in the determinednucleotide sequence region specific to the fungi belonging to the genusByssochlamys, partial regions which satisfy the following fourconditions were searched:

1) including several nucleotides which is specific to the genus;

2) having a GC content of about 30% to 80%;

3) having low possibility to cause self-annealing; and

4) having a Tm value of about 55 to 65° C.

Based on the nucleotide sequences of the above regions, one primer pairwas designed to search the effectiveness of detection of the genusByssochlamys and identification of the genus by PCR reactions using DNAsextracted from the fungi as templates. Specifically, it was examinedthat a DNA amplification reaction is observed at a positioncorresponding to the size of about 150 bp in the case of a reactionusing DNA of the fungi of the genus Byssochlamys as a template, while noamplification product is observed in the cases of reactions usinggenomic DNAs of other fungi as templates. As a result, DNA amplificationwas observed at a position corresponding to the size of about 150 bpspecifically to Byssochlamys nivea and Byssochlamys fulva, while noamplification product was observed in the cases of reactions usinggenomic DNAs of other fungi as templates. The results reveal that it ispossible to exhaustively detect the fungi of the genus Byssochlamys, andto identify the genus Byssochlamys at genus level. The primer pairconfirmed to have the effectiveness is one which consists of theoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 1 and 2. The primers used were synthesized by Sigma-AldrichJapan (desalted products, 0.02 μmol scale) and purchased.

(2) Preparation of Samples

The fungi belonging to the genus Byssochlamys, other heat-resistantfungi, and general fungi shown in Table 1 and Table 2 were used as fungito be used for evaluation of the effectiveness of the designed primers.These fungi were stored in Medical Mycology Research Center (MMRC),Chiba University, and the fungi deposited based on IFM numbers or thelike were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. (for general fungi) or 30° C. (forheat-resistant fungi and Aspergillus fumigatus) for 7 days.

TABLE 1 Sample No. Species Strain No. (IFM) N (Negative Control) — PByssochlamys nivea 48421 1 Aureobasidium pullulans 41408 2 Aureobasidiumpullulans 41409 3 Aureobasidium pullulans 41410 4 Alternaria alternata41348 5 Alternaria alternata 52225 6 Chaetomium globosum 40868 7Chaetomium globosum 40869 8 Chaetomium globosum 40873 9 Paecilomycesvariotii 40913 10 Paecilomyces variotii 50292 11 Paecilomyces variotii40915 12 Trichoderma viride 40938 13 Trichoderma viride 51045 14Cladosporium cladosporioides 41450 15 Fusarium oxysporium 41530 16Fusarium oxysporium 50002 17 Aspergillus fumigatus 07-77 18 Aspergillusfumigatus 07-81 19 Aspergillus fumigatus 07-87 20 Aspergillus fumigatus07-91 21 Aspergillus fumigatus 07-93

TABLE 2 Sample No. Species Strain No.(IFM) 1 Byssochlamys fulva 48421 2Byssochlamys fulva 51213 3 Byssochlamys nivea 51244 4 Byssochlamys nivea51245 5 Talaromyces flavus 42243 6 Talaromyces flavus 52233 7Talaromyces luteus 53241 8 Talaromyces luteus 53242 9 Talaromycestrachyspermus 42247 10 Talaromyces trachyspermus 52252 11 Talaromyceswortmannii 52255 12 Talaromyces wortmannii 52262 13 Neosartorya fischeri46945 14 Neosartorya fischeri 46946 15 Neosartorya glabra 46949 16Neosartorya glabra 46951 17 Neosartorya spinosa 46967 18 Neosartoryaspinosa 46968 19 Neosartorya hiratsukae 46954 20 Neosartorya hiratsukae47036 21 Hamigera avellanea 42323 22 Hamigera avellanea 52241(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (trade name: PrepMan ultra, manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 1 (0.02pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 2 (0.02 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 59° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 3(a) andFIG. 3(b). Note that, FIG. 3(a) shows an electrophoretogram of samplesof the fungi shown in Table 1, and FIG. 3(b) shows an electrophoretogramof samples of the fungi shown in Table 2. The numbers in theelectrophoretograms correspond the sample numbers in the tables, andrepresent samples obtained by using DNAs extracted from the fungi havingthe corresponding sample numbers in the tables.

As a result, in the case of the samples containing the genomic DNA ofthe fungi belonging to the genus Byssochlamys, amplification of genefragments of about 150 bp was confirmed (lanes 1 to 4 in FIG. 3(b)). Onthe other hand, in the case of the samples containing no genomic DNA ofthe fungi belonging to the genus Byssochlamys, amplification of genefragments was not confirmed. From the above-described results, it isunderstood that the fungi belonging to the genus Byssochlamys can bespecifically detected by using the oligonucleotides (a) and (b) of thepresent invention.

(A-2) Detection and Discrimination of Fungi Belonging to the GenusByssochlamys

(1) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 1 and 2, designed inExample 1(A-1), were used.

(2) Preparation of Samples

To confirm detection specificity of the oligonucleotides (a) and (b) tothe fungi belonging to the genus Byssochlamys, the respective strains ofByssochlamys fulva shown in FIG. 4 and the respective strains ofByssochlamys nivea shown in FIG. 5 were used. As the fungi, fungiavailable from fungus deposition institutes, such as fungi stored inNational Institute of Technology and Evaluation based on NBRC numbersand fungi stored in The Centraalbureau voor Schimmelcultures based onCBS numbers were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. for 7 days.

(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (trade name: PrepMan ultra, manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 1 (0.02pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 2 (0.02 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 59° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 4 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 4 and FIG.5. Note that, FIG. 4 shows an electrophoretogram of samples of strainsof Byssochlamys fulva, and FIG. 5 shows an electrophoretogram of sampleso of strains of Byssochlamys nivea.

As a result, in all of the strains of Byssochlamys fulva andByssochlamys nivea used, specific amplified DNA fragments wereconfirmed. Therefore, it is understood that the fungi belonging to thegenus Byssochlamys can be specifically detected with high accuracyregardless of the strains by using the oligonucleotides of the presentinvention.

(B-1) Detection and Discrimination of Fungi Belonging to the GenusTalaromyces

1. Analysis of Nucleotide Sequence Specific to Fungi Belonging to theGenus Talaromyces

Nucleotide sequences of the β-tubulin gene and the ITS region and D1/D2region of 28S rDNA of each variety of fungi belonging to the genusTalaromyces were determined by the following method.

A test fungus was cultured in the dark on a potato dextrose agar slantat 25° C. for 7 days. DNA was extracted from the fungus usingGenTorukun™ (manufactured by TAKARA BIO INC.). PCR amplification of atarget site was performed using PuRe Taq™ Ready-To-Go PCR Beads(manufactured by GE Health Care UK LTD); and primers Bt2a(5′-GGTAACCAAATCGGTGCTGCTTTC-3′, SEQ ID NO: 79) and Bt2b(5′-ACCCTCAGTGTAGTGACCCTTGGC-3′, SEQ ID NO: 80) (Glass and Donaldson,Appl Environ Microbiol 61: 1323-1330, 1995) as primers for the β-tubulingene; or primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′, SEQ ID NO: 81) andNL4 (5′-GGTCCGTGTTTCAAGACGG-3′, SEQ ID NO: 82) (The fungal homorph:Mitotic and plemorphic speciation in fungal systematics, Wallingford:CAB international.) as primers for the D1/D2 region of 28S rDNA.Amplification of β-tubulin partial length was performed under conditionsincluding a denaturation temperature of 95° C., an annealing temperatureof 59° C., an elongation temperature of 72° C., and 35 cycles.Amplification of ITS region and D1/D2 region of 28S rDNA was performedunder conditions including a denaturation temperature of 95° C., anannealing temperature of 55° C., an elongation temperature of 72° C.,and 35 cycles. PCR products were purified using Auto Seg™ G-50(manufactured by Amersham Pharmacia Biotech). The PCR products werelabeled with BigDye (registered trademark) terminator Ver. 1.1(manufactured by Applied Biosystems), and electrophoresis was performedusing ABI PRISM 3130 Genetic Analyzer (manufactured by AppliedBiosystems). Nucleotide sequences from fluorescence signals inelectrophoresis were determined using the software “ATGC Ver. 4”(manufactured by Genetyx).

Based on nucleotide sequence information of the β-tubulin gene and ITSregion and D1/D2 region of 28S rDNA of a variety of fungi (Talaromycesflavus, Talaromyces luteus, and Talaromyces wortmannii) and knownnucleotide sequence information of the β-tubulin gene and ITS region andD1/D2 region of 28S rDNA of a variety of fungi, alignment analyses wereperformed using DNA analysis software (product name: DNAsis pro,manufactured by Hitachi Software Engineering Co., Ltd.), to therebydetermine specific regions in the β-tubulin gene and ITS region andD1/D2 region of 28S rDNA including nucleotide sequences specific to thefungi belonging to the genus Talaromyces (SEQ ID NOS: 26 to 28).

2. Detection of Fungi Belonging to the Genus Talaromyces andIdentification of the Fungi at Genus Level

(1) Design of Primers

From regions having particularly high specificity to the fungi belongingto the genus Talaromyces on the 3′-end side in the determined nucleotidesequence regions specific to the fungi belonging to the genusTalaromyces (SEQ ID NOS: 26 to 28), partial regions which satisfy thefollowing four conditions were searched:

1) including several nucleotides which is specific to the genus;

2) having a GC content of about 30% to 80%;

3) having low possibility to cause self-annealing; and

4) having a Tm value of about 55 to 65° C.

Based on the nucleotide sequences of the above regions, five primerpairs were designed for the β-tubulin gene, and five primer pairs weredesigned for the ITS region and D1/D2 region of 28S rDNA to search theeffectiveness of detection of the fungi of the genus Talaromyces andidentification of the genus by PCR reactions using DNAs extracted fromthe fungi as templates. As a result, in the case of using one of thefive pairs for the β-tubulin gene primers, amplification of DNA wasobserved specifically to Talaromyces flavus and Talaromycestrachyspermus at a position corresponding to the size expected from thedesigned primer pairs. The primer pair confirmed to have theeffectiveness is the pair of SEQ ID NOS: 3 and 4.

Subsequently, it was examined that the fungi of the genus Talaromycescan be exhaustively detected by using a plurality of primer pairs incombination. Specifically, a DNA amplification reaction is observed at aposition corresponding to the size expected from the designed primerpairs in the case of a reaction using DNA of the fungi of the genusTalaromyces as a template, while no amplification product is observed inthe cases of reactions using genomic DNAs of other fungi as templates.As a result, it was confirmed that the fungi of the genus Talaromycescan be exhaustively detected and the genus Talaromyces can be identifiedat genus level by using a mixture of two of the five pairs of theprimers for the β-tubulin gene and two of the five pairs of the primersfor the ITS region and D1/D2 region of 28S rDNA. The primer pairsconfirmed to have the effectiveness are ones each of which consists ofany two oligonucleotides represented by the nucleotide sequences setforth in SEQ ID NOS: 5 to 11. The primers used were synthesized bySigma-Aldrich Japan (desalted products, 0.02 μmol scale) and purchased.

(2) Preparation of Samples

The fungi belonging to the genus Talaromyces, other heat-resistantfungi, and general fungi shown in Table 3 were used as fungi to be usedfor evaluation of the effectiveness of the designed primers. These fungiwere stored in Medical Mycology Research Center (MMRC), ChibaUniversity, and the fungi deposited based on IFM numbers or the likewere obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. (for general fungi) or 30° C. (forheat-resistant fungi) for 7 days.

TABLE 3 Sample No. Species Strain No. N (Negative Control) — 1Talaromyces flavus T38 2 Talaromyces trachyspermus T24 3 Neosartoryaficheri A183 4 Byssochlamys fulva IFM48421 5 Byssochlamys nivea IFM512446 Penicillium griseofulvum P14 7 Penicillium citirinum P15 8 Penicilliumpaneum P16 9 Penicillium oxalicum P17(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 3 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 4 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 95° C. for 10 seconds, (ii) anannealing reaction at 59° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 10 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 6.

As a result, in the case of the samples containing the genomic DNA ofTalaromyces flavus and Talaromyces trachyspermus, amplification of genefragments of about 80 bp was confirmed (lanes 1 and 2 in FIG. 6). On theother hand, in the case of the samples containing no genomic DNA of thefungi belonging to the genus Talaromyces, amplification of genefragments was not confirmed. From the above-described results, it isunderstood that the fungi belonging to the genus Talaromyces can bespecifically detected by using the above-described oligonucleotides (c)and (d).

(B-2) Detection and Discrimination of Fungi Belonging to the GenusTalaromyces

(1) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 5 to 8, designed inExample 1(B-1), were used.

(2) Preparation of Samples

As the fungi belonging to the genus Talaromyces, Talaromyces flavus,Talaromyces trachyspermus, Talaromyces wortmannii, and Talaromycesluteus were used. To confirm specificity of the oligonucleotides (e) to(h) to the β-tubulin genes of the fungi belonging to the genusTalaromyces, the fungi shown in Tables 4 and 5 were used. These fungiwere stored in Medical Mycology Research Center (MMRC), ChibaUniversity, and the fungi deposited based on IFM numbers or the likewere obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. for 7 days.

TABLE 4 Sample No. Species Strain No. 1 Talaromyces flavus 42243 2Talaromyces flavus 52233 3 Talaromyces luteus 53242 4 Talaromyces luteus53241 5 Talaromyces trachyspermus 42247 6 Talaromyces trachyspermus52252 7 Talaromyces wortmannii 52255 8 Talaromyces wortmannii 52262 9Byssochlamys fluva 51213 10 Byssochlamys nivea 51245 11 Hamigeraavellanea 42323 12 Hamigera avellanea 52241

TABLE 5 Sample No. Species Strain No. 1 Talaromyces flavus 42243 2Talaromyces flavus 52233 3 Talaromyces luteus 53242 4 Talaromyces luteus53241 5 Talaromyces trachyspermus 42247 6 Talaromyces trachyspermus52252 7 Talaromyces wortmannii 52255 8 Talaromyces wortmannii 52262 9Alternaria alternata 52225 10 Aureobasidium pullulans 41409 11Chaetomium globosum 40869 12 Hamigera avellanea 42323 13 Paecilomycesvariotii 40913 14 (Negative Control) —(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 25 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 20 μLof sterile distilled water were mixed, and 1.0 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 5 (20pmol/μL), 1.0 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 6 (20 pmol/μL), 1.0 μL of the primer representedby the nucleotide sequence set forth in SEQ ID NO: 7 (20 pmol/μL), and1.0 μL of the primer represented by the nucleotide sequence set forth inSEQ ID NO: 8 (20 pmol/μL) were added thereto, to thereby prepare 50 μLof a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 97° C. for 10 seconds, (ii) anannealing reaction at 59° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 7(a), FIG.7(b) and FIG. 8. Note that, FIG. 7(a) shows an electrophoretogram ofsamples of the fungi shown in Table 4, and FIG. 7(b) shows anelectrophoretogram of samples of the fungi shown in Table 5. The numbersin the electrophoretograms correspond the sample numbers in each table,and represent samples obtained by using DNAs extracted from the fungihaving the corresponding sample numbers in the tables. FIG. 8 shows anelectrophoretogram of samples only of the fungi belonging to the genusTalaromyces.

As a result, in the samples containing genomic DNA of Talaromycesflavus, Talaromyces trachyspermus, or Talaromyces wortmannii of thefungi belonging to the genus Talaromyces (the lanes 1 to 2 and 5 to 8 inFIG. 7(a) and the lanes 1 to 2 and 5 to 8 in FIG. 7(b)), amplificationof gene fragments of about 150 bp was confirmed. The gene fragments wereobtained by amplification using the primers represented by thenucleotide sequences of SEQ ID NOS: 7 and 8. Meanwhile, in the samplescontaining genomic DNA of Talaromyces wortmannii or Talaromyces luteus(the lanes 3 to 4 and 7 to 8 in FIG. 7(a) and the lanes 3 to 4 and 7 to8 in FIG. 7(b)), amplification of gene fragments of about 200 bp wasconfirmed. The gene fragments were obtained by amplification using theprimers represented by the nucleotide sequences of SEQ ID NOS: 5 and 6.On the other hand, in the sample containing no genomic DNA of the fungibelonging to the genus Talaromyces, amplification of gene fragments werenot confirmed. From the above-described results, it is understood thatthe fungi belonging to the genus Talaromyces can be specifically andexhaustively detected by simultaneously using the oligonucleotides (e)and (f) and the oligonucleotides (g) and (h).

(B-3) Detection and Discrimination of Fungi Belonging to the GenusTalaromyces

(1) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 8 and 9, designed inExample 1(B-1), were used.

(2) Preparation of Samples

To confirm detection specificity of the oligonucleotides (h) and (i) tothe fungi belonging to the genus Talaromyces and other fungus shown inFIG. 6 were used. As the fungi, fungi available from fungus depositioninstitutes, such as fungi stored in National Institute of Technology andEvaluation based on NBRC numbers and fungi stored in The Centraalbureauvoor Schimmelcultures based on CBS numbers were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. (for heat-resistant fungi containsthe genus Talaromyces and Aspergillus fumigatus) or 25° C. (for generalfungi) for 7 days.

TABLE 6 Species Strain No. 1 Talaromyces flavus CBS 310. 38 ex type No.2 Talaromyces macrosporus NBRC7132 No. 3 Talaromyces trachysperumusCBS373. 48 ex type No. 4 Talaromyces bacillisporus CBS294. 48 ex typeNo. 5 Talaromyces wortmannii CBS391. 48 ex type No. 6 Talaromyces luteusCBS348. 51 ex neotype No. 7 Geosmithia argillacea CBM-FA0940 ex type No.8 Geosmithia emersonii CBS393. 64 ex type No. 9 Byssochlamys fluvaCBS132. 33 ex type No. 10 Byssochlamys nivea CBS100. 11 ex type No. 11Byssochlamys spectabilis CBS101. 075 ex type No. 12 Hamigera avellaneaCBS295. 48 ex type No. 13 Hamigera striata CBS377. 48 ex type No. 14Thermoascus aurantiacus NBRC6766 No. 15 Thermoascus crustaceus NBRC9129No. 16 Neosartorya fischeri NRRL181 ex type No. 17 Neosartorya spinosaIF08782 ex type No. 18 Aspergillus fumigatus IAM13869 ex type No. 19Aspergillus niger CBS554. 65 ex type No. 20 Aspergillus flavus IF030107ex neotype No. 21 Eupenicillium brefeldianum IF031730 ex type No. 22Penicillium griseofulvum CBS185. 27 ex neotype No. 23 Alternariaalternata CBS103. 33 No. 24 Aurerobasidium pullulans CBS105. 22 No. 25Chaetomium globosum CBS148. 51 No. 26 Fusarium oxysporum IFM50002 No. 27Tricoderma viride CBS433. 34 No. 28 Cladosporium cladosporioides CBS170.54 ex neotype(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 8 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 9 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 59° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 10 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 9-1. Thenumbers in the electrophoretogram correspond the sample numbers in Table6, and represent samples obtained by using DNAs extracted from the fungihaving the corresponding sample numbers in Table 6.

As a result, only in the samples containing genomic DNAs of Talaromycesflavus, Talaromyces macrospores, and Talaromyces trachyspermus of thefungi belonging to the genus Talaromyces (lanes 1 to 3), amplificationof gene fragments of about 150 bp was confirmed. On the other hand, inthe sample containing no genomic DNA of the fungi belonging to the genusTalaromyces, amplification of gene fragments was not confirmed. From theabove-described results, it is understood that only a specific speciesof fungus of the fungi belonging to the genus Talaromyces can bespecifically detected by using the oligonucleotides of the presentinvention.

(B-4) Detection and Discrimination of Fungi Belonging to the GenusTalaromyces

(1) Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 10 and 11, designed inExample 1(B-1), were used.

(2) Preparation of Samples

To confirm detection specificity of the oligonucleotides (j) and (k) tothe same fungi belonging to the genus Talaromyces and other fungus shownin Table 6 of Example 1(B-3) were used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. (for heat-resistant fungi containsthe genus Talaromyces and Aspergillus fumigatus) or 25° C. (for generalfungi) for 7 days.

(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 10 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 11 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 55° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 10 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 9-2. Thenumbers in the electrophoretogram correspond the sample numbers in Table6, and represent samples obtained by using DNAs extracted from the fungihaving the corresponding sample numbers in Table 6.

As a result, only in the samples containing genomic DNAs of Talaromycesbacillisporus, Talaromyces wortmannii, and Talaromyces luteus of thefungi belonging to the genus Talaromyces (lanes 4 to 6), amplificationof gene fragments of about 200 bp was confirmed. On the other hand, inthe sample containing no genomic DNA of the fungi belonging to the genusTalaromyces, amplification of gene fragments was not confirmed. From theabove-described results, it is understood that only a specific speciesof fungus of the fungi belonging to the genus Talaromyces can bespecifically detected by using the oligonucleotides of the presentinvention.

(B-5) Detection and Discrimination of Fungi Belonging to the GenusTalaromyces

(1) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 7 and 8, designed inExample 1(B-1), were used.

(2) Preparation of Samples

To confirm detection specificity to the oligonucleotides (g) and (h)against the fungi belonging to the genus Talaromyces, the strains ofTalaromyces flavus shown in FIG. 10 and strains of Talaromycesmacrosporus shown in FIG. 11 were used. As the fungi, fungi availablefrom fungus deposition institutes, such as fungi stored in NationalInstitute of Technology and Evaluation based on NBRC numbers and fungistored in The Centraalbureau voor Schimmelcultures based on CBS numberswere obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 7 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 8 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 61° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 4 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 10 and FIG.11.

As a result, in the cases of all of the strains of Talaromyces flavusand Talaromyces macrospores used, specific amplified DNA fragments wereconfirmed. Therefore, it is understood that the fungi belonging to thegenus Talaromyces can be specifically detected with high accuracyregardless of the strains by using the oligonucleotides of the presentinvention.

(C-1) Detection and Discrimination of Fungi Belonging to the GenusNeosartorya and Aspergillus fumigatus

1. Determination of Partial Nucleotide Sequence of β-Tubulin Gene

Nucleotide sequences of the β-tubulin gene of Neosartorya glabra,Neosartorya fischeri, Neosartorya spinosa and Aspergillus fumigatus weredetermined by the following method.

A test fungus was cultured in the dark on a potato dextrose agar slantat 30° C. for 7 days. DNA was extracted from the fungus usingGenTorukun™ (manufactured by TAKARA BIO INC.). PCR amplification of atarget site was performed using PuRe Taq™ Ready-To-Go PCR Beads(manufactured by GE Health Care UK LTD); and primers Bt2a(5′-GGTAACCAAATCGGTGCTGCTTTC-3′, SEQ ID NO: 79) and Bt2b(5′-ACCCTCAGTGTAGTGACCCTTGGC-3′, SEQ ID NO: 80) (Glass and Donaldson,Appl Environ Microbiol 61: 1323-1330, 1995). Amplification of β-tubulinpartial length was performed under conditions including a denaturationtemperature of 95° C., an annealing temperature of 59° C., an elongationtemperature of 72° C., and 35 cycles. PCR products were purified usingAuto Seg™ G-50 (manufactured by Amersham Pharmacia Biotech). The PCRproducts were labeled with BigDye (registered trademark) terminator Ver.1.1 (manufactured by Applied Biosystems), and electrophoresis wasperformed using ABI PRISM 3130 Genetic Analyzer (manufactured by AppliedBiosystems). Nucleotide sequences from fluorescence signals inelectrophoresis were determined using the software “ATGC Ver. 4”(manufactured by Genetyx).

Based on the nucleotide sequences of the β-tubulin gene of Neosartoryaglabra, Neosartorya fischeri, Neosartorya spinosa and Aspergillusfumigatus, and known nucleotide sequence information of the β-tubulingene of a variety of fungi, alignment analyses were performed using DNAanalysis software (product name: DNAsis pro, manufactured by HitachiSoftware Engineering Co., Ltd.), to thereby determine specific regionsin the β-tubulin gene including nucleotide sequences specific to thefungi belonging to the genus Neosartorya and Aspergillus fumigatus (SEQID NOS: 32 to 34 and 83 to 86).

2. Detection of Fungi Belonging to the Genus Neosartorya and Aspergillusfumigatus

(1) Design of Primers

From regions having particularly high specificity to the fungi belongingto the genus Neosartorya and Aspergillus fumigatus on the 3′-end side inthe determined nucleotide sequence regions specific to the preservingproperty for the both fungi, partial regions which satisfy the followingfour conditions were searched:

1) including several nucleotides which is specific to the genus;

2) having a GC content of about 30% to 80%;

3) having low possibility to cause self-annealing; and

4) having a Tm value of about 55 to 65° C.

Based on the nucleotide sequences of the above regions, four pairs ofprimers were designed to examine effectiveness of simultaneous detectionof the genus Neosartorya and Aspergillus fumigatus by PCR reactionsusing DNAs extracted from a variety of fungi as templates. Specifically,it was examined that DNA amplification reactions are observed atpositions corresponding to the sizes expected from the designed primerpairs in reactions using DNAs of the genus Neosartorya and Aspergillusfumigatus as templates, while no amplification product is observed inreactions using genomic DNAs of other fungi. As a result, in the casesof two pairs of primers, DNA amplification was observed specifically tothe genus Neosartorya and Aspergillus fumigatus, while, in the cases ofthe reactions using the genomic DNAs of other fungi, no amplificationproduct was observed. That is, the two pairs of primers cansimultaneously detect the genus Neosartorya and Aspergillus fumigatus.The primer pairs confirmed to have the effectiveness are ones each ofwhich consists of any two oligonucleotides represented by the nucleotidesequences set forth in SEQ ID NOS: 12 and 13, and the nucleotidesequences set forth in SEQ ID NOS: 14 and 15. The primers used weresynthesized by Sigma-Aldrich Japan (desalted products, 0.02 μmol scale)and purchased.

(2) Preparation of Samples

The fungi belonging to the genus Neosartorya and Aspergillus fumigatusin Table 7 were used. To confirm specificity of the oligonucleotides (l)and (m) to the β-tubulin genes of these fungi, other fungi shown inTables 7 were used. It should be noted that the strains were obtainedfrom strains stored in RIKEN based on JCM numbers, strains stored inInstitute of Molecular and Cellular Biosciences, The University of Tokyobased on IAM numbers and strains stored in Institute for Fermentation,Osaka based on IFO numbers and used for evaluation.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. (for heat-resistant fungi andAspergillus fumigatus) or 25° C. (for general fungi) for 7 days.

TABLE 7 Sample No. Species Strain No. 1 Neosartorya ficheri A183 2Neosartorya spinosa N121 3 Byssochlamys fulva JCM12805 4 Byssochlamysnivea IAM51244 5 Talaromyces macrosporus IF030070 6 Talaromyces flavusIAM42243 7 Aspergillus fumigatus 07-77 8 Aspergillus niger IF06662 9Aspergillus flavus IF07600 10 Aspergillus terreus IF08835 11 Emericellanidulans IF06083 12 Candida albicans IF01385(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 12 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 13 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 98° C. for 10 seconds, (ii) anannealing reaction at 59° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 10 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 12.

As a result, in the case of the samples containing the genomic DNA ofthe fungi belonging to the genus Neosartorya or Aspergillus fumigatus,amplification of gene fragments of about 100 bp was confirmed (lanes 1,2 and 7). On the other hand, in the case of the samples containing nogenomic DNA of the fungi belonging to the genus Neosartorya orAspergillus fumigatus, amplification of gene fragments was notconfirmed. From the above-described results, it is understood that thefungi belonging to the genus Neosartorya and Aspergillus fumigatus canbe specifically detected by using the above-described oligonucleotides(l) and (m).

(6) Discrimination Based on Difference in Growth Temperatures BetweenFungi Belonging to the Genus Neosartorya and Aspergillus fumigatus

For samples where amplification of gene fragments was confirmed by theabove-mentioned method, hyphae from single colonies were inoculated intoa PDA medium (product name: Potato dextrose medium, manufactured byEiken Chemical Co., Ltd.), and the fungi were cultured at 50° C. for oneday and then observed by a method of confirming hyphae using astereomicroscope. As a result, in samples where Aspergillus fumigatuswas inoculated, active growth of the hyphae was observed, while insamples where the fungi belonging to the genus Neosartorya wereinoculated, growth of the hyphae was not observed. The results revealthat it is possible to discriminate only Aspergillus fumigatus from thefungi belonging to the genus Neosartorya and Aspergillus fumigatus basedon a difference in growth temperature zones.

(C-2) Detection and Discrimination of the Fungi Belonging to the GenusNeosartorya and Aspergillus fumigatus

(1) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 14 and 15, designed inExample 1(C-1), were used.

(2) Preparation of Samples

The fungi belonging to the genus Neosartorya and Aspergillus fumigatusshown in Table 8 and Table 9 were used. To confirm specificity of theoligonucleotides (n) and (o) to the β-tubulin genes of the fungibelonging to the genus Neosartorya and Aspergillus fumigatus, otherfungi shown in Table 8 and Table 9 were used. These fungi were stored inMedical Mycology Research Center (MMRC), Chiba University, and the fungideposited based on numbers were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. (for heat-resistant fungi andAspergillus fumigatus) or 25° C. (for general fungi) for 7 days.

TABLE 8 Sample No. Species Strain No. 1 Neosartorya ficheri A176 2Neosartorya spinosa A176 3 Aspergillus fumigatus A218 4 Neosartoryaglabra N153 5 Neosartorya glabra N129 6 Neosartorya hiratsukae N14 7Aspergillus niger An15 8 Aspergillus terreus A229 9 Aspergillus flavusAs17 10 Emericella nidulans As18 11 N.C. (negative control)

TABLE 9 Sample No. Species Strain No. 1 Neosartorya ficheri A176 2Neosartorya hiratsukae N14 3 Talaromyces luteus T58 4 Talaromyces flavusT38 5 Talaromyces trachyspermus T24 6 Talaromyces wortmannii T77 7Byssochlamys fluva B3 8 Byssochlamys nivea B7 9 Paecilomyces lilacinus54312 10 Penicillium griseofulvum 54313 11 Penicillium citirinum 5431412 Penicillium paneum 55885 13 Penicillium oxalicum 55886 14 N.C.(negative control)(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 14 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 15 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 97° C. for 10 seconds, (ii) anannealing reaction at 59° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 13(a) andFIG. 13(b). Note that, FIG. 13(a) shows an electrophoretogram of samplesof the fungi shown in Table 8, and FIG. 13(b) shows anelectrophoretogram of samples of the fungi shown in Table 9. The numbersin the electrophoretograms correspond the sample numbers in the tables,and represent samples obtained by using DNAs extracted from the fungihaving the corresponding sample numbers in the tables.

As a result, in the case of the samples containing the genomic DNA ofthe fungi belonging to the genus Neosartorya and Aspergillus fumigatus,amplification of gene fragments of about 200 bp was confirmed. On theother hand, in the case of the samples containing no genomic DNA of thefungi belonging to the genus Neosartorya or Aspergillus fumigatus,amplification of gene fragments was not confirmed. From theabove-described results, it is understood that the fungi belonging tothe genus Neosartorya and Aspergillus fumigatus can be specificallydetected by using the above-described oligonucleotides (n) and (o).

(6) Discrimination Based on Difference in Growth Temperatures BetweenFungi Belonging to the Genus Neosartorya and Aspergillus fumigatus

For samples where amplification of gene fragments was confirmed by theabove-mentioned method, hyphae from single colonies were inoculated intoa PDA medium (product name: Potato dextrose medium, manufactured byEiken Chemical Co., Ltd.), and the fungi were cultured at 50° C. for oneday and observed by a method of confirming hyphae using astereomicroscope. As a result, in samples where Aspergillus fumigatuswas inoculated, active growth of the hyphae was observed, while insamples where the fungi belonging to the genus Neosartorya wereinoculated, growth of the hyphae was not observed. The results revealthat it is possible to discriminate only Aspergillus fumigatus from thefungi belonging to the genus Neosartorya and Aspergillus fumigatus basedon a difference in growth temperature zones.

(C-3) Discrimination of Aspergillus fumigatus from Fungi Belonging tothe Genus Neosartorya Using Oligonucleotides (v) and (w)

(1) Design of Primers for Detection of Aspergillus fumigatus

Alignment analyses (DNAsis Pro) of the nucleotide sequences of theβ-tubulin genes of Neosartorya glabra, Aspergillus fumigatus,Neosartorya fischeri, and Neosartorya spinosa represented by SEQ ID NOS:32 to 34 and 83 to 86 were performed to determine regions wheredifferences in nucleotide sequences of both the fungi belonging to thegenus Neosartorya and Aspergillus fumigatus were present. From regionshaving particularly high specificity to Aspergillus fumigatus on the3′-end side in the determined nucleotide sequence regions, partialregions which satisfy the following four conditions were searched:

1) including several nucleotides which is specific to Aspergillusfumigatus;

2) having a GC content of about 30% to 80%;

3) having low possibility to cause self-annealing; and

4) having a Tm value of about 55 to 65° C.

Based on the nucleotide sequences of the above regions, two pairs ofprimers were designed to examine effectiveness of discrimination ofAspergillus fumigatus from the genus Neosartorya and Aspergillusfumigatus by PCR reactions using DNAs extracted from a variety of fungias templates. Specifically, it was examined that, in reactions usingDNAs of Aspergillus fumigatus as templates, DNA amplification reactionsare observed at positions corresponding to the sizes expected from thedesigned primer pairs, while in reactions using genomic DNAs of thefungi belonging to the genus Neosartorya, no amplification product isobserved. As a result, in the cases of one pair of primers, DNAamplification was observed specifically to Aspergillus fumigatus, while,in the cases of the reactions using the genomic DNAs of other fungi, noamplification product was observed. The primer pair confirmed to havethe effectiveness is one each of which consists of any twooligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 22 and 23. The primers used were synthesized bySigma-Aldrich Japan (desalted products, 0.02 μmol scale) and purchased.

(2) Preparation of Samples

The fungi belonging to the genus Neosartorya and Aspergillus fumigatusshown in Table 10 and Table 11 were used. Fungi other than the fungibelonging to the genus Neosartorya and Aspergillus fumigatus, shown inTables 10 and 11, were used as references. These strains of fungi werestored in Medical Mycology Research Center (MMRC), Chiba University, andthe fungi deposited based on numbers were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. (for heat-resistant fungi andAspergillus fumigatus) or 25° C. (for general fungi) for 7 days.

TABLE 10 No. 1 A125 A. fumigatus No. 2 A211 A. fumigatus No. 3 A212 A.fumigatus No. 4 A108 A. fumigatus var. ellipticus No. 5 IFM46945 N.fischeri No. 6 IFM46946 N. fischeri No. 7 IFM46967 N. spinosa No. 8IFM46968 N. spinosa No. 9 IFM46949 N. glabra No. 10 IFM46951 N. glabraNo. 11 IFM46954 N. hiratukae No. 12 IFM47037 N. hiratukae

TABLE 11 No. 1 A209 A. fumigatus No. 2 A213 A. fumigatus No. 3 A215 A.fumigatus No. 4 A176 N. fischeri No. 5 A239 N. fischeri No. 6 A270 N.fischeri No. 7 A178 N. spinosa No. 8 A129 A. brevipes No. 9 A133 A.duricaulis No. 10 A252 A. fumigatiaffinis No. 11 A234 A. fumisynnematusNo. 12 A170 A. lentulus No. 13 A223 A. novofumigatus No. 14 A221 A.udagawae No. 15 A131 A. unilateralis No. 16 A132 A. viridinutaus No. 17An15 A. niger No. 18 A229 A. terreus No. 19 As17 A. flavus No. 20 As18E. nidulans(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 22 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 23 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 59° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2.5 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 14 and FIG.15. Note that, FIG. 14 shows an electrophoretogram of samples of thefungi shown in Table 10, and FIG. 15 shows an electrophoretogram ofsamples of the fungi shown in Table 11. The numbers in theelectrophoretograms correspond the sample numbers in the tables, andrepresent samples obtained by using DNAs extracted from the fungi havingthe corresponding sample numbers in the tables.

As shown in FIGS. 14 and 15, only in the samples containing genomic DNAof Aspergillus fumigatus, amplified fragments of about 200 bp wereclearly confirmed. On the other, hand, in the samples containing genomicDNAs of other fungi including the fungi of the genus Neosartorya, theamplified fragments of about 200 bp were not confirmed.

From the above-described results, it is understood that Aspergillusfumigatus can be specifically detected by performing gene amplificationtreatment using the above-described oligonucleotides (v) and (w) andthen confirming the amplified fragments.

(C-4) Detection and Discrimination of the Fungi Belonging to the GenusNeosartorya and Aspergillus fumigatus

(1) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 14 to 15 and the primersconsisting of the oligonucleotides represented by the nucleotidesequences set forth in SEQ ID NOS: 22 to 23, designed in Example (C−1)and Example (C-3), were used.

(2) Preparation of Samples

To confirm detection specificity of the oligonucleotides (n) and (o) tothe fungi belonging to the genus Neosartorya and detection specificityof the oligonucleotides (v) and (w) to Aspergillus fumigatus, thestrains of Neosartorya fischeri shown in FIG. 16, strains of Neosartoryaglabra shown in FIG. 17, strains of Neosartorya hiratsukae shown in FIG.18, strains of Neosartorya paulistensis shown in FIG. 19, and strains ofNeosartorya spinosa shown in FIG. 20 were used. In addition, Aspergillusfumigatus was used as a positive control for a reaction system includingthe oligonucleotides (v) and (w). It should be noted that, fungiavailable from fungus deposition institutes, such as fungi stored inNational Institute of Technology and Evaluation based on NBRC numbersand fungi stored in The Centraalbureau voor Schimmelcultures based onCBS numbers, and fungi stored in Medical Mycology Research Center, ChibaUniversity based on IFM numbers were obtained and used as test fungi.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 30° C. (for heat-resistant fungi andAspergillus fumigatus) or 25° C. (for general fungi) for 14 days.

(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 14 (0.02pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 15 (0.02 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution. Further, a PCR reactionsolution was prepared in the same way as above except that 0.5 μl of theprimer represented by the nucleotide sequence set forth in SEQ ID NO: 22(20 pmol/μl) and 0.5 μl of the primer represented by the nucleotidesequence set forth in SEQ ID NO: 23 (20 pmol/μl) were used instead ofthe above-mentioned primers.

The PCR reaction solutions were subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 59° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 4 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIG. 16 to FIG.20. Note that, FIG. 16 shows an electrophoretogram of samples of strainsof Neosartorya fischeri fischeri, FIG. 17 shows an electrophoretogram ofsamples of strains of Neosartorya glabra, FIG. 18 shows anelectrophoretogram of samples of strains of Neosartorya hiratsukaefischeri, FIG. 19 shows an electrophoretogram of samples of strains ofNeosartorya paulistensis, and FIG. 20 shows an electrophoretogram ofsamples of strains of Neosartorya spinosa.

As a result, in the reaction systems including the primers representedby the nucleotide sequences set forth in SEQ ID NOS: 14 and 15, specificamplified DNA fragments were confirmed in all of the strains used,Neosartorya fischeri, Neosartorya glabra, Neosartorya hiratsukae,Neosartorya paulistensis, and Neosartorya spinosa (FIG. 16(a), FIG.17(a), FIG. 18(a), FIG. 19(a), and FIG. 20(a)). Therefore, it isunderstood that the fungi belonging to the genus. Neosartorya can bespecifically detected with high accuracy regardless of the strains byusing the oligonucleotides of the present invention.

In the reaction systems including the primers represented by thenucleotide sequences set forth in SEQ ID NOS: 22 and 23, specificamplified DNA fragments were confirmed only in the samples containinggenomic DNA of Aspergillus fumigatus as a template. On the other hand,in all of the samples containing the respective strains of the genusNeosartorya, amplification of DNA fragments was not confirmed (FIG.16(b), FIG. 17(b), FIG. 18(b), FIG. 19(b), and FIG. 20(b)). From theabove-described results, it is understood that Aspergillus fumigatus canbe specifically detected by performing gene amplification treatmentusing the above-described oligonucleotides (v) and (w) and thenconfirming the amplified fragments.

(D-1) Detection and Discrimination of Fungi Belonging to the GenusHamigera

1. Determination of Partial Nucleotide Sequence of β-Tubulin Gene

Nucleotide sequences of the β-tubulin gene of Hamigera avellanea andCladosporium cladosporioides were determined by the following method.

A test fungus was cultured in the dark on a potato dextrose agar slantat 30° C. for Hamigera avellanea and 25° C. for Cladosporiumcladosporioides for 7 days. DNA was extracted from the fungus usingGenTorukun™ (manufactured by TAKARA BIO INC.). PCR amplification of atarget site was performed using PuRe Taq™ Ready-To-Go PCR Beads(manufactured by GE Health Care UK LTD); and primers Bt2a(5′-GGTAACCAAATCGGTGCTGCTTTC-3′, SEQ ID NO: 79) and Bt2b(5′-ACCCTCAGTGTAGTGACCCTTGGC-3′, SEQ ID NO: 80) (Glass and Donaldson,Appl Environ Microbiol 61: 1323-1330, 1995). Amplification of β-tubulinpartial length was performed under conditions including a denaturationtemperature of 95° C., an annealing temperature of 59° C., an elongationtemperature of 72° C., and 35 cycles. PCR products were purified usingAuto Seg™ G-50 (manufactured by Amersham Pharmacia Biotech). The PCRproducts were labeled with BigDye (registered trademark) terminator Ver.1.1 (manufactured by Applied Biosystems), and electrophoresis wasperformed using ABI PRISM 3130 Genetic Analyzer (manufactured by AppliedBiosystems). Nucleotide sequences from fluorescence signals inelectrophoresis were determined using the software “ATGC Ver. 4”(manufactured by Genetyx).

Based on the nucleotide sequences information of the β-tubulin gene ofHamigera avellanea and Cladosporium cladosporioides and known nucleotidesequence information of the β-tubulin gene of a variety of fungi,alignment analyses were performed using DNA analysis software (productname: DNAsis pro, manufactured by Hitachi Software Engineering Co.,Ltd.), to thereby determine specific regions in the β-tubulin geneincluding nucleotide sequences specific to Hamigera avellanea whichbelongs to the genus Hamigera (SEQ ID NOS: 35).

2. Detection of Fungi Belonging to the Genus Hamigera

(1) Design of Primers

From regions having particularly high specificity to Hamigera avellaneaon the 3′-end side in the determined nucleotide sequence regions,partial regions which satisfy the following four conditions weresearched:

1) including several nucleotides which is specific to the genus;

2) having a GC content of about 30% to 80%;

3) having low possibility to cause self-annealing; and

4) having a Tm value of about 55 to 65° C.

Based on the nucleotide sequences of the above regions, five primerpairs were designed to search the effectiveness of detection of thefungi belonging to genus Hamigera by PCR reactions using DNAs extractedfrom the fungi as templates. As a result, in the case of using one ofthe five primer pairs, amplification of DNA was observed specifically toHamigera avellanea and Cladosporium cladosporioides, and noamplification of DNA was observed to genomic DNAs extracted from otherfungi as templates. That is, it was confirmed that the fungi belongingto the genus Hamigera can be detected. The primer pair confirmed to havethe effectiveness is one which consists of the oligonucleotidesrepresented by the nucleotide sequences set forth in SEQ ID NOS: 16 and17. The primers used were synthesized by Sigma-Aldrich Japan (desaltedproducts, 0.02 μmol scale) and purchased.

(2) Preparation of Samples

The fungi belonging to the genus Hamigera, other heat-resistant fungi,and general fungi shown in Table 12 were used as fungi to be used forevaluation of the effectiveness of the designed primers. These fungiwere stored in Medical Mycology Research Center (MMRC), ChibaUniversity, and the fungi deposited based on IFM numbers and T numbersor the like were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. (for general fungi) or 30° C. (forheat-resistant fungi) for 7 days.

TABLE 12 Sample No. Species Strain No. N (Negative Control) — 1 Hamigeraavellanea T34 2 Byssochlamys fulva IAM12805 3 Byssochlamys niveaIAM12806 4 Talaromyces flavus T38 5 Talaromyces trachyspermus T24 6Penicillium griseofulvum P14 7 Penicillium citirinum P15 8 Penicilliumpaneum P16 9 Penicillium oxalicum P17(3) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(4) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 16 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 17 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 98° C. for 10 seconds, (ii) anannealing reaction at 63° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(5) Confirmation of Amplified Gene Fragment

After the PCR reaction, 10 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 21. Thenumbers in the electrophoretogram correspond the sample numbers in Table12, and represent samples obtained by using DNAs extracted from thefungi having the corresponding sample numbers in Table 12.

As a result, in the case of the samples containing the genomic DNA offungi belonging to the genus Hamigera, amplification of gene fragmentsof about 100 bp was confirmed (lane 1). On the other hand, in the caseof the samples containing no genomic DNA of fungi belonging to the genusHamigera, amplification of gene fragments was not confirmed. From theabove-described results, it is understood that fungi belonging to thegenus Hamigera can be specifically detected by using the above-describedoligonucleotides (p) and (q).

(D-2) Detection and Discrimination of Fungi Belonging to the GenusHamigera

(a) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 16 and 17, designed inExample 1(D-1), were used.

(b) Preparation of Samples

As the fungi belonging to the genus Hamigera and the fungi belonging tothe genus Cladosporium, Hamigera avellanea and Cladosporiumcladosporioides shown in Table 13 were used. To confirm specificity ofthe oligonucleotides (p) to (q) to the β-tubulin genes of the fungibelonging to the genus Hamigera and the fungi belonging to the genusCladosporium, other fungi shown in Tables 13 were used. These fungi werestored in Medical Mycology Research Center (MMRC), Chiba University, andthe fungi deposited based on IFM numbers and T numbers or the like wereobtained and used. As a positive control, Hamigera avellanea (the nameof strains: T34) was used as a template of DNA.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. (for general fungi) or 30° C. (forheat-resistant fungi and Aspergillus fumigatus) for 7 days.

TABLE 13 Sample No. Species Strain No. P Hamigera avellanea T34(Positive Control) 1 Hamigera avellanea IAM42323 2 Hamigera avellaneaIAM52241 3 Aureobasidium pullulans IAM41408 4 Aureobasidium pullulansIAM41409 5 Aureobasidium pullulans IAM41410 6 Alternaria alternateIAM41348 7 Alternaria alternate IAM52225 8 Chaetomium globosum IAM408689 Chaetomium globosum IAM4040873 10 Paecilomyces variotii IAM40913 11Paecilomyces variotii IAM40915 12 Paecilomyces variotii IAM50292 13Trichoderma viride IAM40938 14 Trichoderma viride IAM51045 15Cladosporium cladosporioides IAM41450 16 Fusarium oxysporium IAM41530 17Fusarium oxysporium IAM50002 18 Aspergillus fumigatus 07-77 19Aspergillus fumigatus 07-81 20 Aspergillus fumigatus 07-87 21Aspergillus fumigatus 07-91 22 Aspergillus fumigatus 07-93(c) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(d) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 16 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 17 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 97° C. for 10 seconds, (ii) anannealing reaction at 63° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(e) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 22. Thenumbers in the electrophoretogram correspond the sample numbers in Table13, and represent samples obtained by using DNAs extracted from thefungi having the corresponding sample numbers in Table 13.

As a result, in the case of the samples containing the genomic DNA offungi belonging to the genus Hamigera and the fungi belonging to thegenus Cladosporium, amplification of gene fragments of about 100 bp wasconfirmed. On the other hand, in the case of the samples containing nogenomic DNA of the fungi belonging to the genus Hamigera or the fungibelonging to the genus Cladosporium, amplification of gene fragments wasnot confirmed. From the above-described results, it is understood thatthe fungi belonging to the genus Hamigera and the fungi belonging to thegenus Cladosporium can be specifically detected by using theabove-described oligonucleotides (p) and (q).

(D-3) Detection and Discrimination of Fungi Belonging to the GenusHamigera

(a) Design of Primers

Alignment analyses (DNAsis Pro) of nucleotide sequences of the β-tubulingenes of fungi including Cladosporium cladosporioides and Hamigeraavellanea represented by SEQ ID NOS: 35 were performed to determineregions where significant differences in the nucleotide sequences werepresent. From regions having particularly high specificity to Hamigeraavellanea on the 3′-end side in the determined nucleotide sequenceregions, partial regions which satisfy the following four conditionswere searched:

1) including several nucleotides which is specific to the genus;

2) having a GC content of about 30% to 80%;

3) having low possibility to cause self-annealing; and

4) having a Tm value of about 55 to 65° C.

Based on the nucleotide sequences of the above regions, seven pairs ofprimers were designed to examine effectiveness of discrimination of thegenus Hamigera and Cladosporium cladosporioides by PCR reactions usingDNAs extracted from a variety of fungi as templates. Specifically, itwas examined that, in reactions using DNAs of the genus Hamigera astemplates, DNA amplification products are observed at positionscorresponding to the sizes expected from the designed primer pairs,while in reactions using genomic DNAs of the other fungi, noamplification product is observed. As a result, it was confirmed thatthe fungi belonging to the genus Hamigera and Cladosporiumcladosporioides can be detected. The primer pairs confirmed to have theeffectiveness are ones each of which consists of any twooligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 18 and 19, and the nucleotide sequences set forth in SEQ IDNOS: 20 and 21. The primers used were synthesized by Sigma-Aldrich Japan(desalted products, 0.02 μmol scale) and purchased.

(b) Preparation of Samples

The fungi belonging to the genus Hamigera, other heat-resistant fungi,and general fungi shown in Table 14 were used as fungi to be used forevaluation of the effectiveness of the designed primers. These fungiwere stored in Medical Mycology Research Center (MMRC), ChibaUniversity, and the fungi deposited based on IFM numbers or the likewere obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. (for general fungi) or 30° C. (forheat-resistant fungi) for 7 days.

TABLE 14 Sample No. Species Strain No. N (Negative Control) — 1 Hamigeraavellanea T34 2 Hamigera avellanea IAM42323 3 Hamigera avellaneaIAM52241 4 Aureobasidium pullulans IAM41408 5 Aureobasidium pullulansIAM41409 6 Aureobasidium pullulans IAM41410 7 Alternaria alternateIAM41348 8 Alternaria alternate IAM52220 9 Chaetomium globosum IAM4086810 Chaetomium globosum IAM40869 11 Chaetomium globosum IAM40873 12Paecilomyces variotii IAM40913 13 Paecilomyces variotii IAM40915 14Paecilomyces variotii IAM50292 15 Trichoderma viride IAM40938 16Trichoderma viride IAM51045 17 Cladosporium cladosporioides IAM41450 18Fusarium oxysporium IAM41530 19 Fusarium oxysporium IAM50002(c) Preparation of Genomic DNA

The respective fungi were collected from the agar media using platinumloops.

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). The concentration of each DNA solution was adjustedto 50 ng/μL.

(d) PCR Reaction

1 μL of the genomic DNA solution of Hamigera avellanea or Cladosporiumcladosporioides prepared above as a DNA template, 13 μL of Pre Mix Taq(trade name, manufactured by TAKARA BIO INC.) and 10 μL of steriledistilled water were mixed, and 0.5 μL of the primer represented by thenucleotide sequence set forth in SEQ ID NO: 18 (20 pmol/μL) and 0.5 μLof the primer represented by the nucleotide sequence set forth in SEQ IDNO: 19 (20 pmol/μL) were added thereto, to thereby prepare 25 μL of aPCR reaction solution.

The PCR reaction solution was subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 30 cycles of (i) athermal denaturation reaction at 97° C. for 10 seconds, (ii) anannealing reaction at 60° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(e) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 23. Thenumbers in the electrophoretogram correspond the sample numbers in Table14, and represent samples obtained by using DNAs extracted from thefungi having the corresponding sample numbers in Table 14.

As a result, in the case of the samples containing the genomic DNA ofthe fungus belonging to the genus Hamigera, amplification of genefragments of about 200 bp was confirmed (Sample Nos. 1 to 3). On theother hand, in the case of the sample containing the genomic DNA of thefungus belonging to the genus Cladosporium (Sample No. 17) and thesamples containing no genomic DNA of the fungus belonging to the genusHamigera, amplification of gene fragments was not confirmed. As is clearfrom the results, it is understood that the fungi in samples can bediscriminated as the fungi belonging to the genus Hamigera or as thefungi belonging to the genus Cladosporium by using the oligonucleotides(r) and (s).

(D-4) Detection and Discrimination of Fungi Belonging to the GenusHamigera

(a) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 18 to 21, designed inExample 1(D-3), were used.

(b) Preparation of Samples

To confirm specificity of the oligonucleotides (r) to (u) to theβ-tubulin genes of the fungi belonging to the genus Hamigera, eachstrains of Hamigera striata shown in FIG. 24-1 were used as the fungibelonging to the genus Hamigera.

The respective fungi were cultured in the same way as in (D-1) above.

(c) Preparation of Genomic DNA

Genomic DNA solutions were prepared in the same way as in (D-1) above.The concentration of each of the DNA solutions was adjusted to 50 ng/μl.

(d) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 18 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 19 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution. Further, a PCR reactionsolution was prepared in the same way as above except that 0.5 μl of theprimer represented by the nucleotide sequence set forth in SEQ ID NO: 20(20 pmol/μl) and 0.5 μl of the primer represented by the nucleotidesequence set forth in SEQ ID NO: 21 (20 pmol/μl) were used instead ofthe above-mentioned primers.

The PCR reaction solutions were subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 61° C. to 59° C. for 1 minute, and (iii) an elongationreaction at 72° C. for 1 minute.

(e) Confirmation of Amplified Gene Fragment

After the PCR reaction, 4 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 24-1.

As a result, in either case of the reaction systems including theprimers represented by the nucleotide sequences set forth in SEQ ID NOS:18 and 19 and the primers represented by the nucleotide sequences setforth in SEQ ID NOS: 20 and 21, specific amplified DNA fragments wereconfirmed in all of the used strains of Hamigera striata (lanes 9 to16). The bands were detected more clearly (lanes 9 to 12) in the casesof the reaction system including the primers represented by thenucleotide sequences set forth in SEQ ID NOS: 20 and 21. From theabove-described results, it is understood that the fungi belonging tothe genus Hamigera can be specifically detected with high accuracyregardless of the strains by using the oligonucleotides of the presentinvention.

(D-5) Detection and Discrimination of Fungi Belonging to the GenusHamigera

(a) Preparation of Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 18 to 21, designed inExample 1(D-3), were used.

(b) Preparation of Samples

To confirm specificity of the oligonucleotides (r) to (u) to theβ-tubulin genes of the fungi belonging to the genus Hamigera, eachstrains of Hamigera avellanea shown in FIG. 24-2 were used as the fungibelonging to the genus Hamigera.

The respective fungi were cultured in the same way as in (D-1) above.

(c) Preparation of Genomic DNA

Genomic DNA solutions were prepared in the same way as in (D-1) above.The concentration of each of the DNA solutions was adjusted to 50 ng/μl.

(d) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 18 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 19 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution. Further, a PCR reactionsolution was prepared in the same way as above except that 0.5 μl of theprimer represented by the nucleotide sequence set forth in SEQ ID NO: 20(20 pmol/μl) and 0.5 μl of the primer represented by the nucleotidesequence set forth in SEQ ID NO: 21 (20 pmol/μl) were used instead ofthe above-mentioned primers.

The PCR reaction solutions were subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 59° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(e) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretogram in the agarose gel is shown in FIG. 24-2.

As a result, in either case of the reaction systems including theprimers represented by the nucleotide sequences set forth in SEQ ID NOS:18 and 19 and the primers represented by the nucleotide sequences setforth in SEQ ID NOS: 20 and 21, specific amplified DNA fragments wereconfirmed in all of the used strains of Hamigera avellanea (lanes 1 to8). From the above-described results, it is understood that the fungibelonging to the genus Hamigera can be specifically detected with highaccuracy regardless of the strains by using the oligonucleotides of thepresent invention.

(D-6) Detection and Discrimination of Fungi Belonging to the GenusHamigera

(a) Primers

The primers consisting of the oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 18 to 21, designed inExample 1(D-3), were used.

(b) Preparation of Samples

To confirm specificity of the oligonucleotides (r) to (u) to the fungibelonging to the genus Hamigera, each strains of Byssochlamys nivea andByssochlamys fulva shown in FIGS. 25-1 and 25-2 were used as the fungiof the genus Byssochlamys closely related to the genus Hamigera. As thefungi, fungi available from fungus deposition institutes, such as fungistored in National Institute of Technology and Evaluation based on NBRCnumbers and fungi stored in The Centraalbureau voor Schimmelculturesbased on CBS numbers were obtained and used.

The respective fungi were cultured in the same way as in Example 1(D-1)above.

(c) Preparation of Genomic DNA

Genomic DNA solutions were prepared in the same way as in Example 1(D-1)above. The concentration of each of the DNA solutions was adjusted to 50ng/μl.

(d) PCR Reaction

1 μL of the genomic DNA solution prepared above as a DNA template, 13 μLof Pre Mix Taq (trade name, manufactured by TAKARA BIO INC.) and 10 μLof sterile distilled water were mixed, and 0.5 μL of the primerrepresented by the nucleotide sequence set forth in SEQ ID NO: 18 (20pmol/μL) and 0.5 μL of the primer represented by the nucleotide sequenceset forth in SEQ ID NO: 19 (20 pmol/μL) were added thereto, to therebyprepare 25 μL of a PCR reaction solution. Further, a PCR reactionsolution was prepared in the same way as above except that 0.5 μl of theprimer represented by the nucleotide sequence set forth in SEQ ID NO: 20(20 pmol/μl) and 0.5 μl of the primer represented by the nucleotidesequence set forth in SEQ ID NO: 21 (20 pmol/μl) were used instead ofthe above-mentioned primers.

The PCR reaction solutions were subjected to a gene amplificationtreatment using an automatic gene amplification device thermal cyclerDICE (TAKARA BIO INC.). PCR reaction conditions were 35 cycles of (i) athermal denaturation reaction at 95° C. for 1 minute, (ii) an annealingreaction at 59° C. for 1 minute, and (iii) an elongation reaction at 72°C. for 1 minute.

(e) Confirmation of Amplified Gene Fragment

After the PCR reaction, 2 μL of a sample was collected from the PCRreaction solution and electrophoresed using a 2% agarose gel, and DNAwas stained with SYBR Safe DNA gel stain in 1×TAE (Invitrogen), tothereby confirm whether the amplified DNA fragment was present or not.The electrophoretograms in the agarose gel are shown in FIGS. 25-1 and25-2.

As shown in FIG. 25-1, in the reaction system including the primersrepresented by the nucleotide sequences set forth in SEQ ID NOS: 18 and19, gene amplification was observed at a position corresponding to thesize of 200 bp not only in the case of Hamigera avellanea used as apositive control but also in the cases of part of the strains ofByssochlamys fulva (NBRC31877 and NBRC31878; lanes 9 and 10), while geneamplification was not observed in the cases of the other fungi belongingto the genus Byssochlamys. It is presumed that gene amplification ofNBRC31877 and 31878 was observed because the strains are geneticallyrelated to the genus Hamigera compared with the other strains.

On the other hand, as shown in FIG. 25-2, in the reaction systemincluding the primers represented by the nucleotide sequences set forthin SEQ ID NOS: 20 and 21, gene amplification was not observed inByssochlamys fulva NBRC31877 and NBRC31878. From the above-describedresults, it is understood that only the fungi belonging to the genusHamigera can be specifically detected with high accuracy withoutdetecting the fungi belonging to the genus Byssochlamys by using theoligonucleotides set forth in SEQ ID NOS: 20 and 21.

As the above, it is understood that the heat-resistant fungi can bedetected by using the oligonucleotides of the present invention.Specifically, the fungi belonging to the genus Byssochlamys can bediscriminated by using the oligonucleotides of SEQ ID NOS: 1 and 2. Thefungi belonging to the genus Talaromyces can be discriminated by usingthe oligonucleotides SEQ ID NOS: 3 to 11. The fungi belonging to thegenus Neosartorya and Aspergillus fumigatus can be discriminated byusing the oligonucleotides SEQ ID NOS: 12 to 15. Further, the fungibelonging to the genus Neosartorya can be discriminated from Aspergillusfumigatus. The fungi belonging to the genus Hamigera can bediscriminated by using the oligonucleotides SEQ ID NOS: 16 to 21.Therefore, it is possible to discriminate the heat-resistant fungi byperforming at least two, preferably all of the steps of detecting theheat-resistant fungi using the above oligonucleotides of the presentinvention.

Example 2: Detection of Fungi Belonging to the Genus Byssochlamys

(1) Design and Synthesis of Primers

Nucleotide sequence information of the ITS region and D1/D2 region of28S rDNA of a variety of fungi (Paecilomyces variotii, Hamigeraavellanea, Talaromyces flavus, Talaromyces luteus, Talaromycestrachyspermus, Byssochlamys nivea, Byssochlamys fulva, and Neosartoryafischeri) was determined by a sequencing method. Based on the sequenceinformation, alignment analyses were performed using DNA analysissoftware (product name: DNAsis pro, manufactured by Hitachi SoftwareEngineering Co., Ltd.), to thereby determine nucleotide sequencesspecific to the fungi belonging to the genus Byssochlamys. Based on thespecified nucleotide sequence, primers consisting of oligonucleotidesrepresented by the nucleotide sequences set forth in SEQ ID NOS: 36 to39 were designed, and the primers were synthesized by E Genome order(FUJITSU SYSTEM SOLUTIONS LIMITED) (SEQ ID NOS: 36 and 37; 5 pmol scale,SEQ ID NOS: 38 and 39; 40 pmol scale; all of the primers arecolumn-purified products) and purchased.

(2) Preparation of Samples

As the fungi belonging to the genus Byssochlamys, Byssochlamys fulva andByssochlamys nivea were used. To confirm the specificity of the primersconsisting of oligonucleotides represented by the nucleotide sequencesof SEQ ID NOS: 36 to 39 to the ITS region and D1/D2 region of 28S rDNAof the fungi belonging to the genus Byssochlamys, the fungi shown inTable 15 were used. These fungi were stored in Medical Mycology ResearchCenter (MMRC), Chiba University, and the fungi deposited based on IFMnumbers or the like were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 15 Sample No. Species Strain No. (IFM) 1 Byssochlamys fulva 484212 Byssochlamys nivea 51244 3 Talaromyces flavus 42243 4 Talaromycesluteus 53241 5 Talaromyces trachyspermus 42247 6 Talaromyces wortmannii52262 7 Neosartorya ficheri 46945 8 Neosartorya spinosa 46967 9Neosartorya glabra 46949 10 Neosartorya hiratsukae 47036 11 Alternariaalternata 41348 12 Aureobasidium pullulans 41409 13 Chaetomium globosum40869 14 Fusarium oxysporium 50002 15 Trichoderma viride 40938 16Cladosporium cladosporioides 41450(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 36 (LB1F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 37 (LB1B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 38(LB1FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 39 (LB1BIP primer: 40 pmol/μL), 1 μL of Bst DNA Polymerase (8U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and 1 μL of thetemplate DNA prepared above were mixed, and distilled water was addedthereto, to thereby prepare a total of 25 μL of a reaction solution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 35(a) and FIG. 35(b). Note that,FIG. 35(a) shows the results of samples Nos. 1 to 8 in Table 15, andFIG. 35(b) shows the results of samples Nos. 9 to 16 in Table 15.

As a result, the turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 30 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofthe fungi belonging to the genus Byssochlamys were used as templates.The increase in the turbidity reached a peak 60 to 70 minutes after thestart of the reaction, and then the turbidity was in a gradual decline.

On the other hand, in the systems where the genomic DNAs of the fungiother than the genus Byssochlamys were used, the turbidity increases inthe reaction solutions were not observed for 90 minutes after theinitiation of the reaction. It should be noted that, in the systemsincluding genomic DNAs of fungi other than the genus Byssochlamys, theturbidity increases in the reaction solutions were observed from about100 minutes after the start of the reaction. This is caused byamplification by reactions of the primers or annealing of a small amountof primers to sequences other than the target sequences due to a longerreaction time.

As is apparent from the above results, according to the presentinvention, it is possible to detect the genus Byssochlamys easily,rapidly, and specifically.

Example 3: Detection of Fungi Belonging to the Genus Neosartorya andAspergillus fumigatus

(1) Design and Synthesis of Primers

Nucleotide sequence information of the β-tubulin genes of a variety offungi (Neosartorya fischeri, Neosartorya glabra, Paecilomyces variotii,Hamigera avellanea, Talaromyces flavus, Talaromyces luteus, Talaromycestrachyspermus, Byssochlamys nivea, and Byssochlamys fulva) wasdetermined by a sequencing method. Based on the sequence information,alignment analyses were performed using DNA analysis software (productname: DNAsis pro, manufactured by Hitachi Software Engineering Co.,Ltd.), to thereby determine nucleotide sequences specific to the fungibelonging to the genus Neosartorya and Aspergillus fumigatus. Based onthe nucleotide sequences, primers consisting of oligonucleotidesrepresented by the nucleotide sequences set forth in SEQ ID NOS: 40 to45 were designed, and the primers were synthesized by E Genome order(FUJITSU SYSTEM SOLUTIONS LIMITED) (SEQ ID NOS: 40 and 41; 5 pmol scale,SEQ ID NOS: 42 and 43; 40 pmol scale, SEQ ID NOS: 44 and 45: 20 pmolscale; all of the primers are column-purified products) and purchased.

(2) Preparation of Samples

The fungi belonging to the genus Neosartorya and Aspergillus fumigatusshown in Tables 16 and 16-1 were used. To confirm the specificity of theprimers consisting of oligonucleotides represented by the nucleotidesequences of SEQ ID NOS: 40 to 45 to the β-tubulin genes of the fungi,the fungi shown in Tables 16 and 16-1 were used. These fungi were storedin Medical Mycology Research Center (MMRC), Chiba University, and thefungi deposited based on IFM numbers or the like were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 16 Sample No. Species Strain No. (IFM) 1 Neosartorya ficheri 469452 Neosartorya spinosa 46967 3 Neosartorya glabra 46949 4 Neosartoryahiratsukae 47036 5 Talaromyces flavus 42243 6 Talaromyces luteus 53242 7Talaromyces trachyspermus 42247 8 Talaromyces wortmannii 52262 9Byssochlamys fulva 48421 10 Hamigera avellanea 42323 11 Alternariaalternata 41348 12 Aureobasidium pullulans 41409 13 Chaetomium globosum40869 14 Fusarium oxysporium 50002 15 Trichoderma viride 40938 16Cladosporium cladosporioides 41450

TABLE 16-1 Sample No. Species Strain No. 1 Neosartorya ficheri IFM469462 Neosartorya ficheri IFM46945 3 Neosartorya ficheri A176 4 Neosartoryaspinosa IFM46968 5 Neosartorya spinosa IFM46967 6 Neosartorya spinosaA178 7 Neosartorya glabra IFM46949 8 Neosartorya glabra IFM46951 9Neosartorya hiratsukae IFM46954 10 Neosartorya hiratsukae IFM47036 11Aspergillus fumigatus A218 12 Aspergillus niger An15 13 Aspergillusterreus A229 14 Aspergillus flavus As17 15 Emericella nidulans As18 16NC (Negative Control) DW(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 40 (LN1F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 41 (LN1B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 42(LN1FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 43 (LN1BIP primer: 40 pmol/μL), 1 μL of the primer consisting ofthe oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 44 (LN1LF loop primer: 20 pmol/μL), 1 μL of the primerconsisting of the oligonucleotide represented by the nucleotide sequenceset forth in SEQ ID NO: 45 (LN1LB loop primer: 20 pmol/μL), 1 μL of BstDNA Polymerase (8 U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and1 μL of the template DNA prepared above were mixed, and distilled waterwas added thereto, to thereby prepare a total of 25 μL of a reactionsolution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 36 and FIG. 36-1.

As a result, the turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 20 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofthe fungi belonging to the genus Neosartorya (Neosartorya spinosa,Neosartorya hiratsukae, Neosartorya fischeri, and Neosartorya glabra)and Aspergillus fumigatus were used as templates.

On the other hand, in the systems where the genomic DNAs of the fungiother than the genus Neosartorya and Aspergillus fumigatus were used,the turbidity increases in the reaction solutions were not observed for50 minutes after the initiation of the reaction. It should be notedthat, in the systems including genomic DNAs of the fungi other than thegenus Neosartorya and Aspergillus fumigatus, the turbidity increases inthe reaction solutions were observed from about 60 minutes after thestart of the reaction. This is caused by amplification by reactions ofthe primers or annealing of a small amount of primers to sequences otherthan the target sequences due to a longer reaction time.

As is apparent from the above results, according to the presentinvention, it is possible to detect the fungi belonging to the genusNeosartorya and Aspergillus fumigatus easily, rapidly, and specifically.

Example 4: Detection of Fungi Belonging to the Genus Hamigera

(1) Design and Synthesis of Primers

Nucleotide sequence information of the β-tubulin genes of a variety offungi (Paecilomyces variotii, Hamigera avellanea, Talaromyces flavus,Talaromyces luteus, Talaromyces trachyspermus, Byssochlamys nivea,Byssochlamys fulva, and Neosartorya fischeri) was determined by asequencing method. Based on the sequence information, alignment analyseswere performed using DNA analysis software (product name: DNAsis pro,manufactured by Hitachi Software Engineering Co., Ltd.), to therebydetermine nucleotide sequences specific to the fungi belonging to thegenus Hamigera. Based on the specific nucleotide sequence, primersconsisting of oligonucleotides represented by the nucleotide sequencesset forth in SEQ ID NOS: 51 to 56 were designed, and the primers weresynthesized by E Genome order (FUJITSU SYSTEM SOLUTIONS LIMITED) (SEQ IDNOS: 51 and 52; 5 pmol scale, SEQ ID NOS: 53 and 54; 40 pmol scale, SEQID NOS: 55 and 56: 20 pmol scale; all of the primers are column-purifiedproducts) and purchased.

(2) Preparation of Samples

As the fungi belonging to the genus Hamigera, Hamigera avellanea shownin Table 17 were used. To confirm the specificity of the primersconsisting of oligonucleotides represented by the nucleotide sequencesof SEQ ID NOS: 51 to 56 to the β-tubulin genes of the fungi belonging tothe genus Hamigera, the other fungi shown in Table 17 were also used.These fungi were stored in Medical Mycology Research Center (MMRC),Chiba University, and the fungi deposited based on IFM numbers or thelike were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 17 Sample No. Species Strain No. (IFM) 1 Hamigera avellanea 423232 Hamigera avellanea 52241 3 Hamigera avellanea 52957 4 Byssochlamysfulva 51213 5 Byssochlamys nivea 51245 6 Paecilomyces variotii 40913 7Paecilomyces variotii 40915 8 DW (Negative Control) —(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 51 (LH2F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 52 (LH2B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 53(LH2FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 54 (LH2BIP primer: 40 pmol/μL), 1 μL of the primer consisting ofthe oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 55 (LH2LF loop primer: 20 pmol/μL), 1 μL of the primerconsisting of the oligonucleotide represented by the nucleotide sequenceset forth in SEQ ID NO: 56 (LH2LB loop primer: 20 pmol/μL), 1 μL of BstDNA Polymerase (8 U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and1 μL of the template DNA prepared above were mixed, and distilled waterwas added thereto, to thereby prepare a total of 25 μL of a reactionsolution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 37.

As a result, the turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 25 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofthe fungi belonging to the genus Hamigera (Hamigera avellanea) were usedas templates.

On the other hand, in the systems where the genomic DNAs of the fungiother than the fungi belonging to the genus Hamigera were used, theturbidity increases in the reaction solutions were not observed for 100minutes after the initiation of the reaction. It should be noted that,in the systems including genomic DNAs of the fungi other than the genusHamigera, the turbidity increases in the reaction solutions wereobserved from about 110 minutes after the start of the reaction. This iscaused by amplification by reactions of the primers or annealing of asmall amount of primers to sequences other than the target sequences dueto a longer reaction time.

As is apparent from the above results, according to the presentinvention, it is possible to detect the fungi belonging to the genusHamigera easily, rapidly, and specifically.

Example 5: Detection Aspergillus fumigatus (Discrimination Aspergillusfumigatus from the Fungi Belonging to the Genus Neosartorya)

(1) Design and Synthesis of Primers

Nucleotide sequence information of the β-tubulin genes of a variety offungi (Aspergillus fumigatus, Neosartorya fischeri, and Neosartoryaspinosa) was determined by a sequencing method. Based on the sequenceinformation, alignment analyses were performed using DNA analysissoftware (product name: DNAsis pro, manufactured by Hitachi SoftwareEngineering Co., Ltd.), to thereby determine nucleotide sequencesspecific to Aspergillus fumigatus. Based on the specified nucleotidesequence, primers consisting of oligonucleotides represented by thenucleotide sequences set forth in SEQ ID NOS: 46 to 50 were designed,and the primers were synthesized by E Genome order (FUJITSU SYSTEMSOLUTIONS LIMITED) (SEQ ID NOS: 46 and 47; 5 pmol scale, SEQ ID NOS: 48and 49; 40 pmol scale, SEQ ID NO: 50: 20 pmol scale; all of the primersare column-purified products) and purchased.

(2) Preparation of Samples

The fungi belonging to the genus Neosartorya and Aspergillus fumigatusshown in Table 18 were used. These fungi were stored in Medical MycologyResearch Center (MMRC), Chiba University, and the fungi deposited basedon IFM numbers or the like were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 18 Sample No. Species Strain No. 1 Aspergillus fumigatus A209 2Aspergillus fumigatus A213 3 Aspergillus fumigatus A215 4 Neosartoryaficheri IFM46945 5 Neosartorya ficheri IFM46946 6 Neosartorya spinosaIFM46967 7 Neosartorya spinosa IFM46968 8 DW (Negative Control) —(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 46 (LAf2F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 47 (LAf2B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 48(LAf2FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 49 (LAf2BIP primer: 40 pmol/μL), 1 μL of the primer consisting ofthe oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 50 (LAf2LB loop primer: 20 pmol/μL), 1 μL of Bst DNAPolymerase (8 U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and 1μL of the template DNA prepared above were mixed, and distilled waterwas added thereto, to thereby prepare a total of 25 μL of a reactionsolution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 38.

As a result, the turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 45 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofAspergillus fumigatus were used as templates.

On the other hand, in the systems where the genomic DNAs of the fungibelonging to the genus Neosartorya were used, the turbidity increases inthe reaction solutions were not observed for 90 minutes after theinitiation of the reaction. It should be noted that, in the systemsincluding genomic DNAs of the fungi belonging to the genus Neosartorya,the turbidity increases in the reaction solutions were observed fromabout 100 minutes after the start of the reaction. This is caused byamplification by reactions of the primers or annealing of a small amountof primers to sequences other than the target sequences due to a longerreaction time.

As is apparent from the above results, according to the presentinvention, it is possible to detect Aspergillus fumigatus easily,rapidly, and specifically.

In addition, it is possible to discrimination the fungi belonging to thegenus Neosartorya from Aspergillus fumigatus by utilizing the method ofdetecting Aspergillus fumigatus shown in the present Example and themethod of using primers consisting of oligonucleotides represented bythe nucleotide sequences set forth in SEQ ID NOS: 40 to 45 shown inExample 3.

Example 6: Detection of Talaromyces flavus

(1) Design and Synthesis of Primers

Nucleotide sequence information of the β-tubulin genes of a variety offungi (Paecilomyces variotii, Hamigera avellanea, Talaromyces flavus,Talaromyces luteus, Talaromyces trachyspermus, Byssochlamys nivea,Byssochlamys fulva, and Neosartorya fischeri) was determined by asequencing method. Based on the sequence information, alignment analyseswere performed using DNA analysis software (product name: DNAsis pro,manufactured by Hitachi Software Engineering Co., Ltd.), to therebydetermine nucleotide sequences specific to Talaromyces flavus. Based onthe specified nucleotide sequence, primers consisting ofoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 57 to 61 were designed, and the primers were synthesized byE Genome order (FUJITSU SYSTEM SOLUTIONS LIMITED) (SEQ ID NOS: 57 and58; 5 pmol scale, SEQ ID NOS: 59 and 60; 40 pmol scale, SEQ ID NO: 61:20 pmol scale; all of the primers are column-purified products) andpurchased.

(2) Preparation of Samples

Talaromyces flavus shown in Table 19 were used. To confirm thespecificity of the primers consisting of oligonucleotides represented bythe nucleotide sequences of SEQ ID NOS: 57 to 61 to the β-tubulin genesof Talaromyces flavus, the other fungi shown in Table 19 were used.These fungi were stored in Medical Mycology Research Center (MMRC),Chiba University, and the fungi deposited based on IFM numbers or thelike were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 19 Sample No. Species Strain No. (IFM) 1 Talaromyces flavus 422432 Talaromyces flavus 52233 3 Talaromyces luteus 53242 4 Talaromycesluteus 53241 5 Talaromyces trachyspermus 42247 6 Talaromycestrachyspermus 52252 7 Talaromyces wortmannii 52255 8 Talaromyceswortmannii 52262 9 Byssochlamys fulva 48421 10 Byssochlamys fluva 5121311 Byssochlamys nivea 51244 12 Byssochlamys nivea 51245 13 Hamigeraavellanea 42323 14 Hamigera avellanea 52241 15 Paecilomyces variotii40913 16 Paecilomyces variotii 40915(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 57 (LTf2F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 58 (LTf2B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 59(LTf2FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 60 (LTf2BIP primer: 40 pmol/μL), 1 μL of the primer consisting ofthe oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 61 (LTf2LB loop primer: 20 pmol/μL), 1 μL of Bst DNAPolymerase (8 U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and 1μL of the template DNA prepared above were mixed, and distilled waterwas added thereto, to thereby prepare a total of 25 μL of a reactionsolution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 39(a) and FIG. 39(b). Note that,FIG. 39(a) shows the results of samples Nos. 1 to 8 in Table 19, andFIG. 39(b) shows the results of samples Nos. 9 to 16 in Table 19.

As a result, the turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 30 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofTalaromyces flavus were used as templates.

On the other hand, in the systems where the genomic DNAs of the fungiother than Talaromyces flavus were used, the turbidity increases in thereaction solutions were not observed for 70 minutes after the initiationof the reaction. It should be noted that, in the systems includinggenomic DNAs of the fungi other than Talaromyces flavus, increases inthe turbidity of the reaction solutions were observed from about 80minutes after the start of the reaction. This is caused by amplificationby reactions of the primers or annealing of a small amount of primers tosequences other than the target sequences due to a longer reaction time.

As is apparent from the above results, according to the method of thepresent invention, it is possible to easily and rapidly detectTalaromyces flavus by measuring the turbidity in a reaction solution fora period from the start of the reaction to the time point of about 60minutes, at which the turbidity significantly increases by DNAamplification of only Talaromyces flavus.

Example 7: Detection of Talaromyces wortmannii

(1) Design and Synthesis of Primers

Nucleotide sequence information of the β-tubulin genes of a variety offungi (Talaromyces wortmannii, Paecilomyces variotii, Hamigeraavellanea, Talaromyces flavus, Talaromyces luteus, Talaromycestrachyspermus, Byssochlamys nivea, Byssochlamys fulva, and Neosartoryafischeri) was determined by a sequencing method. Based on the sequenceinformation, alignment analyses were performed using DNA analysissoftware (product name: DNAsis pro, manufactured by Hitachi SoftwareEngineering Co., Ltd.), to thereby determine nucleotide sequencesspecific to Talaromyces wortmannii. Based on the specified nucleotidesequence regions, primers consisting of oligonucleotides represented bythe nucleotide sequences set forth in SEQ ID NOS: 62 to 67 weredesigned, and the primers were synthesized by E Genome order (FUJITSUSYSTEM SOLUTIONS LIMITED) (SEQ ID NOS: 62 and 63; 5 pmol scale, SEQ IDNOS: 64 and 65; 40 pmol scale, SEQ ID NOS: 66 and 67: 20 pmol scale; allof the primers are column-purified products) and purchased.

(2) Preparation of Samples

Talaromyces wortmannii shown in Table 20 were used. To confirm thespecificity of the primers consisting of oligonucleotides represented bythe nucleotide sequences of SEQ ID NOS: 62 to 67 to the β-tubulin genesof Talaromyces wortmannii, the other fungi shown in Table 20 were used.These fungi were stored in Medical Mycology Research Center (MMRC),Chiba University, and the fungi deposited based on IFM numbers or thelike were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 20 Sample No. Species Strain No. (IFM) 1 Talaromyces wortmannii52255 2 Talaromyces wortmannii 52262 3 Talaromyces flavus 42243 4Talaromyces luteus 53241 5 Talaromyces trachyspermus 42247 6Byssochlamys fulva 48421 7 Byssochlamys nivea 51244 8 Hamigera avellanea42323(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 62 (LTw4F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 63 (LTw3B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 64(LTw4FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 65 (LTw3BIP primer: 40 pmol/μL), 1 μL of the primer consisting ofthe oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 66 (LTw4LF loop primer: 20 pmol/μL), 1 μL of the primerconsisting of the oligonucleotide represented by the nucleotide sequenceset forth in SEQ ID NO: 67 (LTw3LB loop primer: 20 pmol/μL), 1 μL of BstDNA Polymerase (8 U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and1 μL of the template DNA prepared above were mixed, and distilled waterwas added thereto, to thereby prepare a total of 25 μL of a reactionsolution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 40.

As a result, the turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 20 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofTalaromyces wortmannii were used as templates.

On the other hand, in the systems where the genomic DNAs of the fungiother than Talaromyces wortmannii were used, the turbidity increases inthe reaction solutions were not observed for 40 minutes after theinitiation of the reaction. It should be noted that, in the systemsincluding genomic DNAs of the fungi other than Talaromyces wortmannii,increases in the turbidity of the reaction solutions were observed fromabout 50 minutes after the start of the reaction. This is caused byamplification by reactions of the primers or annealing of a small amountof primers to sequences other than the target sequences due to a longerreaction time.

As is apparent from the above results, according to the presentinvention, it is possible to detect Talaromyces wortmannii easily,rapidly, and specifically.

Example 8: Detection of Talaromyces luteus

(1) Design and Synthesis of Primers

Nucleotide sequence information of the β-tubulin genes of a variety offungi (Talaromyces luteus, Paecilomyces variotii, Hamigera avellanea,Talaromyces flavus, Talaromyces wortmannii, Talaromyces trachyspermus,Byssochlamys nivea, Byssochlamys fulva, and Neosartorya fischeri) wasdetermined by a sequencing method. Based on the sequence information,alignment analyses were performed using DNA analysis software (productname: DNAsis pro, manufactured by Hitachi Software Engineering Co.,Ltd.), to thereby determine nucleotide sequences specific to Talaromycesluteus. Based on the specified nucleotide sequence, primers consistingof oligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 68 to 72 were designed, and the primers were synthesized byE Genome order (FUJITSU SYSTEM SOLUTIONS LIMITED) (SEQ ID NOS: 68 and69; 5 pmol scale, SEQ ID NOS: 70 and 71; 40 pmol scale, SEQ ID NO: 72:20 pmol scale; all of the primers are column-purified products) andpurchased.

(2) Preparation of Samples

Talaromyces luteus shown in Table 21 were used. To confirm thespecificity of the primers consisting of oligonucleotides represented bythe nucleotide sequences of SEQ ID NOS: 68 to 72 to the β-tubulin genesof Talaromyces luteus, the other fungi shown in Table 21 were used.These fungi were stored in Medical Mycology Research Center (MMRC),Chiba University, and the fungi deposited based on IFM numbers or thelike were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 21 Sample No. Species Strain No. (IFM) 1 Talaromyces luteus 532422 Talaromyces luteus 53241 3 Talaromyces flavus 42243 4 Talaromycestrachyspermus 42247 5 Talaromyces wortmannii 52262 6 Byssochlamys fulva48421 7 Neosartorya ficheri 46945 8 Neosartorya spinosa 46967 9Neosartorya glabra 46949 10 Neosartorya hiratsukae 47036 11 Alternariaalternata 41348 12 Aureobasidium pullulans 41409 13 Chaetomium globosum40869 14 Fusarium oxysporium 50002 15 Trichoderma viride 40938 16Cladosporium cladosporioides 41450(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 68 (LTI1F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 69 (LTI1B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 70(LTI1FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 71 (LTI1BIP primer: 40 pmol/μL), 1 μL of the primer consisting ofthe oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 72 (LTI1LF loop primer: 20 pmol/μL), 1 μL of Bst DNAPolymerase (8 U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and 1μL of the template DNA prepared above were mixed, and distilled waterwas added thereto, to thereby prepare a total of 25 μL of a reactionsolution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 41(a) and FIG. 41(b). Note that,FIG. 41(a) shows the results of samples Nos. 1 to 8 in Table 21, andFIG. 41(b) shows the results of samples Nos. 9 to 16 in Table 21.

As a result, the sudden turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 25 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofTalaromyces luteus were used as templates.

On the other hand, in the systems where the genomic DNAs of the fungiother than Talaromyces luteus were used, the turbidity increases in thereaction solutions were not observed for 80 minutes after the initiationof the reaction. It should be noted that, in the systems includinggenomic DNAs of the fungi other than Talaromyces luteus, increases inthe turbidity of the reaction solutions were observed from about 90minutes after the start of the reaction. This is caused by amplificationby reactions of the primers or annealing of a small amount of primers tosequences other than the target sequences due to a longer reaction time.Although a gradual increase in the turbidity was observed from 10minutes after the start of the reaction in the sample number 8, theincrease is considered to be caused not by amplification of the genecorresponding to the nucleotide sequence specific to the genomic DNA butby gradual gene amplification by a reaction of the primers. The gradualgene amplification reaction can be clearly differentiated from theconventional LAMP reaction, because the measurement results between theboth reactions are clearly different. That is, the LAMP reaction showsthe results obtained by amplification caused by annealing of primers,while the gradual gene amplification shows the results having no peak ofthe turbidity increase.

As is apparent from the above results, according to the presentinvention, it is possible to identify Talaromyces luteus easily,rapidly, and specifically.

Example 9: Detection of Talaromyces flavus and Talaromyces Trachyspermus

(1) Design and Synthesis of Primers

Nucleotide sequence information of the ITS region and D1/D2 region of28S rDNA of a variety of fungi (Talaromyces flavus, Talaromycestrachyspermus, Paecilomyces variotii, Hamigera avellanea, Talaromyceswortmannii, Byssochlamys nivea, Byssochlamys fulva, and Neosartoryafischeri) was determined by a sequencing method. Based on the sequenceinformation, alignment analyses were performed using DNA analysissoftware (product name: DNAsis pro, manufactured by Hitachi SoftwareEngineering Co., Ltd.), to thereby determine nucleotide sequencesspecific to Talaromyces flavus and Talaromyces trachyspermus. Based onthe specified nucleotide sequence, primers consisting ofoligonucleotides represented by the nucleotide sequences set forth inSEQ ID NOS: 73 to 78 were designed, and the primers were synthesized byE Genome order (FUJITSU SYSTEM SOLUTIONS LIMITED) (SEQ ID NOS: 73 and74; 5 pmol scale, SEQ ID NOS: 75 and 76; 40 pmol scale, SEQ ID NOS: 77and 78; 20 pmol scale; all of the primers are column-purified products)and purchased.

(2) Preparation of Samples

Talaromyces flavus and Talaromyces trachyspermus shown in Table 22 wereused. To confirm the specificity of the primers consisting ofoligonucleotides represented by the nucleotide sequences of SEQ ID NOS:73 to 78 to the ITS region and D1/D2 region of 28S rDNA of the fungi,the other fungi shown in Table 22 were used. These fungi were stored inMedical Mycology Research Center (MMRC), Chiba University, and the fungideposited based on IFM numbers or the like were obtained and used.

The respective fungi were cultured under optimum conditions. The culturewas performed using a potato dextrose medium (trade name: Pearlcorepotato dextrose agar medium, manufactured by Eiken Chemical Co., Ltd.)under culture conditions of 25° C. for 7 days.

TABLE 22 Sample No. Species Strain No. 1 Talaromyces flavus IFM42243 2Talaromyces flavus IFM52233 3 Talaromyces flavus T38 4 Talaromycesluteus IFM53242 5 Talaromyces luteus IFM53241 6 Talaromycestrachyspermus IFM42247 7 Talaromyces trachyspermus IFM52252 8Talaromyces wortmannii IFM52262 9 Talaromyces wortmannii IFM52255 10Byssochlamys fulva IFM48421 11 Byssochlamys nivea IFM51245 12Penicillium griseofulvum IFM54313 13 Penicillium citirinum IFM54314 14Hamigera avellanea IFM42323 15 Neosartorya ficheri IFM46945 16 NC(Negative Control) DW(3) Preparation of Genomic DNA

Genomic DNA solutions were prepared from the collected fungi using agenomic DNA preparation kit (PrepMan ultra (trade name) manufactured byApplied Biosystems). Specifically, several colonies were collected fromeach medium, and the fungus was suspended in 200 μL of a reagentsupplied with the kit and dissolved by a heat treatment at 100° C. for10 minutes. Centrifugation was performed at 14,800 rpm for 5 minutes,and the supernatant was collected. The concentration of the resultantgenomic DNA solution was adjusted to 50 ng/μL. The genomic DNA solutionwas used as a template DNA in the following LAMP reaction.

(4) Preparation of Reaction Solution for LAMP Reaction

12.5 μL of 2× Reaction Mix (Tris-HCl (pH 8.8) 40 mM, KCl 20 mM, MgSO₄ 16mM, (NH₄)₂SO₄ 20 mM, 0.2% Tween20, Betaine 1.6 M, dNTPs 2.8 mM: EikenChemical Co., Ltd.; Loopamp DNA amplification reagent kit), 1 μL of theprimer consisting of the oligonucleotide represented by the nucleotidesequence set forth in SEQ ID NO: 73 (LT1F3 primer: 5 pmol/μL), 1 μL ofthe primer consisting of the oligonucleotide represented by thenucleotide sequence set forth in SEQ ID NO: 74 (LT1B3 primer: 5pmol/μL), 1 μL of the primer consisting of the oligonucleotiderepresented by the nucleotide sequence set forth in SEQ ID NO: 75(LT1FIP primer: 40 pmol/μL), 1 μL of the primer consisting of theoligonucleotide represented by the nucleotide sequence set forth in SEQID NO: 76 (LT1BIP primer: 40 pmol/μL), 1 μL of the primer consisting ofthe oligonucleotide represented by the nucleotide sequence set forth inSEQ ID NO: 77 (LT1LF primer: 20 pmol/μL), 1 μL of the primer consistingof the oligonucleotide represented by the nucleotide sequence set forthin SEQ ID NO: 78 (LT1LB loop primer: 20 pmol/μL), 1 μL of Bst DNAPolymerase (8 U/25 μL, manufactured by Eiken Chemical Co., Ltd.) and 1μL of the template DNA prepared above were mixed, and distilled waterwas added thereto, to thereby prepare a total of 25 μL of a reactionsolution.

(5) LAMP Reaction

The reaction solution prepared above was subjected to a DNAamplification reaction at 63±2° C. for 60 minutes using a real-timeturbidity measuring apparatus Loopamp RT-160C (manufactured by EikenChemical Co., Ltd.). Simultaneously, the turbidity of the reactionsolution was measured (wavelength: 400 nm).

(6) Confirmation of DNA Amplification

Amplification of DNA was confirmed by an increase in turbidity of thereaction solution. The measurement results of the turbidity of thereaction solutions are shown in FIG. 42.

As a result, the turbidity increases (i.e. the DNA synthesis andamplification reactions) were observed from about 40 minutes after theinitiation of the reaction only in the systems where the genomic DNAs ofTalaromyces flavus and Talaromyces trachyspermus were used as templates.

On the other hand, in the systems where the genomic DNAs of the fungiother than Talaromyces flavus and Talaromyces trachyspermus were used,the turbidity increases in the reaction solutions were not observed for45 minutes after the initiation of the reaction. It should be notedthat, in the systems including genomic DNAs of the fungi other thanTalaromyces flavus and Talaromyces trachyspermus, increases in theturbidity of the reaction solutions were observed from about 50 minutesafter the start of the reaction. This is caused by amplification byreactions of the primers or annealing of a small amount of primers tosequences other than the target sequences due to a longer reaction time.

As is apparent from the above results, according to the presentinvention, it is possible to detect Talaromyces flavus and Talaromycestrachyspermus easily, rapidly, and specifically.

As shown in Examples 6 to 9, it is possible to detect the fungi of thegenus Talaromyces at species level according to the method of thepresent invention.

Having described our invention as related to the present embodiments, itis our intention that the invention not be limited by any of the detailsof the description, unless otherwise specified, but rather be construedbroadly within its spirit and scope as set out in the accompanyingclaims.

This non-provisional application claims priority under 35 U.S.C. § 119(a) on Patent Application No. 2008-139995 filed in Japan on May 28,2008, Patent Application No. 2008-139996 filed in Japan on May 28, 2008,Patent Application No. 2008-139997 filed in Japan on May 28, 2008,Patent Application No. 2008-139998 filed in Japan on May 28, 2008,Patent Application No. 2008-139999 filed in Japan on May 28, 2008, andPatent Application No. 2008-141499 filed in Japan on May 29, 2008, eachof which is entirely herein incorporated by reference.

The invention claimed is:
 1. A method of detecting whether aheat-resistant fungus is present in a sample, wherein the heat-resistantfungus is a member of the genus Talaromyces, the method comprising thesteps of: (i) adding, to the sample, or adding to nucleic acid obtainedfrom the sample, a primer set selected from the group consisting ofprimer set no. 1, 2, 5 and 6, wherein: primer set no. 1 consists of apair of oligonucleotides that consists of oligonucleotide (a), whichconsists of a labeled or unlabeled nucleotide sequence that has 95% ormore homology to SEQ ID NO: 10; and oligonucleotide (b), which consistsof a labeled or unlabeled nucleotide sequence that has 95% or morehomology to SEQ ID NO: 11; primer set no. 2 consists of a group ofoligonucleotides that consists of oligonucleotide (a), which consists ofa labeled or unlabeled nucleotide sequence that has 95% or more homologyto SEQ ID NO: 7; oligonucleotide (a′), which consists of a labeled orunlabeled nucleotide sequence that has 95% or more homology to SEQ IDNO: 9; and oligonucleotide (b), which consists of a labeled or unlabelednucleotide sequence that has 95% or more homology to SEQ ID NO: 8;primer set no. 5 consists of a pair of oligonucleotides that consists ofoligonucleotide (a), which consists of a labeled or unlabeled nucleotidesequence that has 95% or more homology to SEQ ID NO: 3; andoligonucleotide (b), which consists of a labeled or unlabeled nucleotidesequence that has 95% or more homology to SEQ ID NO: 4; and primer setno. 6 consists of a pair of oligonucleotides that consists ofoligonucleotide (a), which consists of a labeled or unlabeled nucleotidesequence that has 95% or more homology to SEQ ID NO: 5; andoligonucleotide (b), which consists of a labeled or unlabeled nucleotidesequence that has 95% or more homology to SEQ ID NO: 6; (ii) hybridizingthe oligonucleotide pair or group in the primer set to nucleic acid inthe sample or to nucleic acid obtained from the sample, under conditionsin which the oligonucleotides in the primer set hybridize specificallyto the nucleic acid of a member of the genus Talaromyces that is in thesample or was obtained from the sample, or, hybridizing theoligonucleotide pair or group in the primer set to nucleic acid in thesample or to nucleic acid obtained from the sample, under conditions inwhich the oligonucleotides in the primer set hybridize specifically tonucleic acid of a member of the genus Talaromyces that is in the sampleor was obtained from the sample and performing amplification of thenucleic acid using the oligonucleotides in the primer set as primers forthe amplification, and (iii) determining whether the oligonucleotides inthe primer set hybridized to nucleic acid in step (ii), or, determiningwhether an amplification product was produced in step (ii), wherein,determining that the oligonucleotides in primer set no. 1, 2, 5 or 6hybridized to the nucleic acid, or, when gene amplification wasperformed using the oligonucleotides in primer set no. 1, 2, 5 or 6,determining that an amplification product was produced by theamplification, detects the presence, in the sample, of a fungusbelonging to the genus Talaromyces.
 2. The method of detecting aheat-resistant fungus according to claim 1, wherein at least oneoligonucleotide is labeled.
 3. The method of claim 1, wherein the primerset is primer set no. 1, the nucleotide sequence of oligonucleotide (a)is that of SEQ ID NO: 10 and the nucleotide sequence of oligonucleotide(b) is that of SEQ ID NO:
 11. 4. The method of claim 1, wherein theprimer set is primer set no. 2, the nucleotide sequence ofoligonucleotide (a) is that of SEQ ID NO: 7, the nucleotide sequence ofoligonucleotide (a′) is that of SEQ ID NO: 9 and the nucleotide sequenceof oligonucleotide (b) is that of SEQ ID NO:
 8. 5. The method of claim1, wherein the primer set is primer set no. 5, the nucleotide sequenceof oligonucleotide (a) is that of SEQ ID NO: 3 and the nucleotidesequence of oligonucleotide (b) is that of SEQ ID NO:
 4. 6. The methodof claim 1, wherein the primer set is primer set no. 6, the nucleotidesequence of oligonucleotide (a) is that of SEQ ID NO: 5 and thenucleotide sequence of oligonucleotide (b) is that of SEQ ID NO:
 6. 7.The method of claim 1, wherein the fungus is Talaromyces flavus,Talaromyces trachyspermus, Talaromyces luteus, Talaromyces wortmannii,Talaromyces bacillisporus or Talaromyces macrosporus.
 8. The methodaccording to claim 1, wherein step (ii) is hybridizing theoligonucleotides in the primer set to nucleic acid in the sample or tonucleic acid obtained from the sample, and step (iii) is deteiminingwhether the oligonucleotides in the primer set hybridized to nucleicacid in step (ii).
 9. The method of claim 1, wherein step (ii) ishybridizing the oligonucleotides in the primer set to nucleic acid inthe sample or to nucleic acid obtained from the sample and performingamplification of the nucleic acid using the oligonucleotides in theprimer set as primers for the amplification and step (iii) isdetermining whether an amplification product was produced in step (ii).